Cell2015_Preview_Its a SMAD-SMAD World-metastatic prediction

欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁氉氉氉氉引文格式：曾澳，闫语，王淑荣，张妍，何宇茜．Ｓｍａｄ蛋白调控角膜新生血管发生发展的研究进展［Ｊ］．眼科新进展，２０２２，４２（１）：７５ ７８．ｄｏｉ：１０．１３３８９／ｊ．ｃｎｋｉ．ｒａｏ．２０２２．００１７【文献综述】Ｓｍａｄ蛋白调控角膜新生血管发生发展的研究进展△曾　澳　闫　语　王淑荣　张　妍　何宇茜欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁氉氉氉氉作者简介：曾澳（ＯＲＣＩＤ：００００ ０００１６２６８ ６９５８），男，１９９９年１１月出生，湖南娄底人。

主要研究方向：角膜病。

Ｅ ｍａｉｌ：１０１８７７５０２９＠ｑｑ．ｃｏｍ通信作者：何宇茜（ＯＲＣＩＤ：００００ ０００１ ５８０２ ８０６３），女，１９８９年７月出生，吉林松原人，博士。

主要研究方向：角膜病。

Ｅ ｍａｉｌ：ｈｅｙｕｘｉｈｏｔ＠１６３．ｃｏｍ收稿日期：２０２１ ０１ ２２修回日期：２０２１ ０９ ３０本文编辑：盛丽娜△基金项目：吉林省科技厅国际科技合作项目（编号：２０２００８０１０１６ＧＨ）；吉林省科技厅自然科学基金（编号：２０１８０１０１１４６ＪＣ）作者单位：１３００４１　吉林省长春市，吉林大学第二医院眼科中心（曾澳，闫语，王淑荣，张妍，何宇茜）；１３００２１　吉林省长春市，吉林大学白求恩医学部（曾澳，闫语）【摘要】　角膜新生血管（ＣＮＶ）是一种严重的致盲性病理改变，与多种眼表疾病的发生发展密切相关。

在ＣＮＶ发生发展过程中，多种蛋白参与调控。

研究表明，Ｓｍａｄ蛋白可通过多种信号通路影响ＣＮＶ的发生发展。

本文就近年来Ｓｍａｄ蛋白调控ＣＮＶ发生发展的研究进展作一综述。

【关键词】　Ｓｍａｄ蛋白；角膜新生血管；转化生长因子 β；骨形态发生蛋白；茎细胞；尖细胞【中图分类号】　Ｒ７７２．２角膜受到不利因素的刺激时，可通过产生角膜新生血管（ＣＮＶ）来加强角膜的免疫防御功能，并且促进角膜的愈合。

682017.08基础研究TGF-β1、Smad3在星形细胞瘤肿瘤组织中的表达及其相关性马志君1　吉　洁2　李春辉3　王维兴11承德医学院附属医院神经外科 河北省承德市 067000 2承德医学院附属医院病案室 河北省承德市 0670003承德医学院附属医院病理科 河北省承德市 067000【摘　要】目的：探讨TGF-β1、Smad3在星形细胞瘤肿瘤组织中的表达情况以及两者的相关性。

方法：采用免疫组化染色检测30例手术切除的星形细胞瘤患者肿瘤组织标本中TGF-β1和Smad3的表达水平，并对两者的相关性进行分析。

结果：TGF-β1和Smad3表达水平在不同级别星形细胞瘤中各组间均有显著差异（P<0.01），TGF-β1的表达与Smad3表达水平之间呈明显正相关。

结论：TGF-β1和Smad3在星形细胞瘤肿瘤组织中的表达水平与肿瘤的病理分级呈正相关，TGF-β1/Smad3信号传导通路在星形细胞瘤的异常增殖过程中起到了重要作用。

【关键词】星形细胞瘤；TGF-β1；Smad3星形细胞瘤约占颅脑肿瘤的13%-26%，其主要的生物学行为是恶性增殖和侵袭 [1]。

目前手术仍然是临床治疗的主要方法，但术后肿瘤复发率高，预后差成为治疗该病的难点。

因此从分子生物学的角度来探讨星形细胞瘤增殖、侵袭的机制，为靶向治疗提供理论依据具有重要意义。

本实验以手术切除的星形细胞瘤患者肿瘤组织为研究标本，分别检测低级别星形细胞瘤和高级别星形细胞瘤中TGF-β1和Smad3表达情况，从分子生物学的角度研究其在星形细胞瘤发病机制中的影响，使其成为临床治疗中新的靶点。

1 材料与方法1.1 材料与试剂男性18例、女性12例，年龄12~76岁，平均（56.25±7.51）岁；参照WHO 分型标准：低级别星形细胞瘤（Ⅰ~Ⅱ级）14例，高级别星形细胞瘤（Ⅲ~Ⅳ级）16例。

兔抗人TGF-β1、Smad3单克隆抗体（美国Cell Signaling 公司）；浓缩型DAB 试剂盒（北京中山金桥公司）；免疫组化MaxVision 试剂盒（上海拜力生物科技公司）；Trizol 试剂盒（上海联硕生物科技公司）。

气道平滑肌在气道重塑中作用的研究进展史丽;强丽霞;金寿德【摘要】哮喘和慢性阻塞性肺疾病是临床常见的慢性呼吸系统疾病.在长期炎症等因素刺激下气道出现上皮损伤、上皮网状基膜增厚,黏液细胞化生,气道平滑肌细胞发生增生、肥大、迁移和凋亡抑制等改变,使气道发生结构重塑,最终导致肺功能下降.其中平滑肌细胞的相关改变在气道重塑中起重要作用,阐明气道平滑肌在气道重塑中的作用机制是目前防治哮喘和慢性阻塞性肺疾病的重要课题.本文综述气道平滑肌在气道重塑中主要作用与相关机制及气道重塑对哮喘和慢性阻塞性肺疾病的影响.【期刊名称】《医学研究杂志》【年(卷),期】2018(047)011【总页数】4页(P202-204,208)【关键词】气道平滑肌;气道重塑;哮喘;慢性阻塞性肺疾病【作者】史丽;强丽霞;金寿德【作者单位】150001 哈尔滨医科大学附属第四医院;150001 哈尔滨医科大学附属第四医院;150001 哈尔滨医科大学附属第四医院【正文语种】中文【中图分类】R562哮喘(asthma)[1]和慢性阻塞性肺疾病(chronic obstructive pulmonarydisease,COPD) (简称慢阻肺)[2]是呼吸系统常见的慢性气道疾病。

气道重塑是两者主要发病机制之一。

尽管诊疗水平不断提高，哮喘和慢阻肺的发生率与病死率并未明显下降，尤其是发生气道重塑的这部分人群，其治疗难度更大、病死率更高。

研究发现，气道平滑肌(airway smooth muscle，ASM)在气道重塑过程中发挥着至关重要的作用。

目前大量研究表明，气道平滑肌细胞发生增生、肥大、迁移和凋亡抑制，同时表达和释放细胞因子、生长因子以及蛋白酶类可以导致上皮下基膜增厚和细胞外基质(extracellular matrixc，ECM)沉积，引起气道重塑，是最终引起肺功能下降的主要原因。

本文就气道平滑肌在气道重塑中的作用及气道重塑对哮喘和慢阻肺的影响做一综述。

BMP4和SMAD4基因在公绵羊繁殖相关组织的表达采复拉·大木拉;田志龙;段新华;储明星【摘要】试验旨在探索骨形态发生蛋白4(Bone morphogenetic protein4,BMP4)和SMAD家族蛋白4(SMAD family member 4,SMAD4)基因在公绵羊繁殖中的作用,以便深入研究公绵羊繁殖机制.采用荧光定量PCR技术检测BMP4和SMAD4基因在高繁殖力的小尾寒羊公羊(n=3)和低繁殖力苏尼特公羊(n=3)大脑、小脑、下丘脑、垂体、输精管、肾上腺、睾丸和附睾8种组织中的表达.结果表明:BMP4和SMAD4基因在小尾寒羊公羊和苏尼特羊公羊各种组织中均有表达.其中BMP4基因在睾丸中高表达,在输精管、肾上腺中中等表达,在其他组织中均呈痕量表达;SMAD4基因在睾丸、下丘脑、小脑、大脑中高表达,在其他组织中中等表达.BMP4基因在低繁殖力苏尼特羊公羊睾丸中表达量显著高于高繁殖力小尾寒羊公羊(P<0.05);SMAD4基因在低繁殖力苏尼特公羊8种组织中表达量均显著高于高繁殖力小尾寒羊公羊(P<0.05).说明BMP4和SMAD4基因在公绵羊繁殖中起到一定程度的负调控作用,这为公羊高繁殖性状选育提供了参考依据.【期刊名称】《中国草食动物科学》【年(卷),期】2019(039)002【总页数】4页(P5-8)【关键词】BMP4基因;SMAD4基因;公绵羊;组织表达【作者】采复拉·大木拉;田志龙;段新华;储明星【作者单位】中国农业科学院北京畜牧兽医研究所,农业部动物遗传育种与繁殖重点实验室,北京 100193;新疆畜牧科学院畜牧业质量标准研究所,乌鲁木齐 830000;中国农业科学院北京畜牧兽医研究所,农业部动物遗传育种与繁殖重点实验室,北京100193;河南科技大学动物科技学院,洛阳 471003;新疆畜牧科学院畜牧业质量标准研究所,乌鲁木齐 830000;中国农业科学院北京畜牧兽医研究所,农业部动物遗传育种与繁殖重点实验室,北京 100193【正文语种】中文【中图分类】S826.2转化生长因子β（Transforming growth factor-β，TGF-β）超家族可以调节动物细胞的增殖、分化、凋亡，影响动物的胚胎发育、器官形成以及细胞外基质合成和稳态［1］。

肝纤维化是肝炎-肝硬化-肝癌三部曲的重要病理表型，研究表明抑制肝纤维化能有效减缓肝炎向肝癌的进程［1］。

但目前临床上针对肝纤维化没有行之有效的治疗方案［2］。

因而，深入探索纤维化机制为寻找延缓乃至逆转纤维化的治疗靶点和节约医疗资源具有十分重要意义。

肝巨噬细胞在肝纤维化中发挥至关重要的作用，其主要功能与炎症、肝细胞损伤、肝星状细胞的活化和纤维化密切相关［3］。

肝星状细胞在肝脏生理和纤维生成起到关键作用，其能够受到肝巨噬细胞的调控［4］。

近来Lipopolysaccharide stimulates macrophages to secrete exosomes containing miR-155-5p to promote activation and migration of hepatic stellate cellsLIN Jiayi 1,LOU Anni 1,LI Xu 1,21Department of Emergency Medicine,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;2Key Laboratory of First Aid and Trauma Research,Ministry of Education,Hainan Medical College,Haikou 571199,China摘要：目的探索脂多糖（LPS ）刺激下的巨噬细胞来源的外泌体对肝星状细胞的激活及迁移能力的影响及分子机制。

方法以100ng/mL 丙二醇甲醚醋酸（PMA ）处理人THP-1巨噬细胞24h ，诱导其分化为巨噬细胞，给予脂多糖刺激后收集巨噬细胞的培养上清，运用超速离心法提取外泌体并加以鉴定。

荧光定量PCR （qRT-PCR ）检测外泌体中miR-155-5p 的表达。

采用Transwell 共培养体系观察巨噬细胞分泌的外泌体对肝星状细胞LX2增殖、氧化应激、迁移和I 型胶原等纤维化标志物表达的影响。

Molecular Biology Organization (Short-Term Fellowship,J.M.S.),the Koch Institute for Integrative Cancer Research (Graduate Fellowship,S.L.K.),the Deutsche Forschungsgemeinschaft (GK1772,SPP1395,N.B.),the Bundesministerium für Bildung und Forschung (FORSYS,BCCN A5,N.B.),Harvard Medical School institutionalOnderzoek (NWO)(Vici award,A.v.O.),and the European Research Council (grant ERC-AdG 294325-GeneNoiseControl,A.v.O.).SUPPLEMENTARY MATERIALS/content/348/6230/128/suppl/DC1Figs.S1to S16References27October 2014;accepted 11February 2015that H3K9methylation and heterochromatin can 1323APRIL 2015•VOL 348ISSUE SCIENCERESEARCH |REPORTSWellcome Trust Centre for Cell Biology and Institute of Cell o n O c t o b e r 13, 2015w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mheterochromatin nucleation,spreading,and main-tenance (13–15).The inheritance of heterochromatin on centromere repeat DNA inserted at ectopic locations also requires RNAi,but at the mating-type locus heterochromatin is dependent on DNA binding factors in the absence of RNAi (13–17).However,constitutive tethering of Clr4to a euch-romatic locus via the Gal4DNA binding domain (GBD)allows the assembly of an extensive domain of H3K9me-heterochromatin,independently of RNAi (18).In this study,we tethered a regu-latable TetR off -Clr4fusion protein to deter-mine whether H3K9me is a persistent histone modification that can be stably copied through mitotic cell divisions and meiosis after release or loss of the TetR off -Clr4initiator.TetR off -2xFLAG-Clr4-cdd fusion protein (abbre-domain,was stably expressed in cells with an ade6+gene downstream of 4xtetO binding sites at the ura4locus (4xtetO-ade6+)(Fig.1A)(19).TetR-Clr4*silences 4xtetO-ade6+independently of RNAi (ago1D ,dcr1D ),similar to GBD-Clr4(18),resulting in reduced RNA polymerase II associ-ation and high H3K9me2levels and silencing over a broad region (Fig.1C and figs.S1and S9,C to E).TetR-Clr4*is released within 5min from tetO sites by addition of anhydrotetracycline (AHT)(Fig.1B).All strains used in these experiments also express wild-type (WT)Clr4,which can interact via its chromodomain with TetR-Clr4–directed H3K9me and thus potentially use its read-write capabilities to methylate newly incorporated H3and allow heterochromatin transmission to daughter cells after TetR-Clr4*release.However,4xtetO-ade6+after AHT addition;>90%is lost within 6hours (Fig.1C and fig.S2A).AHT itself does not affect endogenous heterochromatin integrity (fig.S2B).H3levels do not decline on 4xtetO-ade6+over this period (fig.S2A).Swi6HP1is also lost from 4xtetO-ade6+when cells are grown with AHT (fig.S1F).We also tethered TetR-Clr4*within two non-essential genes with long open reading frames,which are less likely to contain unannotated fea-tures that might interfere with heterochromatin integrity.Moreover,both sib1+(15,005base pairs)and vps1302+(9200base pairs)exhibit expres-sion levels and rates of H3turnover that are ~three times lower than those of ade6+(Fig.2,A to C,and fig.S3).4xtetO and 1xtetO sites were placed within sib1and vps1203,respectively (Fig.SCIENCE 3APRIL 2015•VOL 348ISSUE 6230133RESEARCH |REPORTSalso placed under the control of low,medium (med ),and high versions of the constitutive adh1promoter (20).We also generated sib1:4xtetO and vps1302:1xtetO without promoters (no ).All strains expressed WT Clr4and TetR-Clr4*.Both sib1+and vps1302+were expressed at low levels when their promoters were removed and at much higher levels from med-adh1or high-adh1compared with their own or low-adh1promoters (fig.S4).TetR-Clr4*was unable to establish much H3K9me2when tethered to sib1:4xtetO or vps1302:1xtetO expressed from hi-adh1and relatively low levels when expressed from med-adh1,but substantial H3K9me2occurred when either gene had no ,its own ,or the low-adh1promoter (fig.S4).How-ever,as with 4xtetO-ade6+,rapid loss of H3K9me2followed TetR-Clr4*release from even no and own promoter constructs;again,>90%was lost within 6hours (Fig.2,D and E).Although high levels of transcription across tethering sites prevents the establishment of H3K9me by TetR-Clr4*,neither low promoter strength nor low H3turnover renders H3K9me more persis-tent upon methyltransferase release.Thus,the inability to maintain H3K9methylation upon re-moval of the initiating tethered Clr4methyltrans-ferase is probably a general feature of euchromatic loci.To determine whether the loss of H3K9meth-ylation from the tethering site is coupled to replication or passage through the cell cycle,we released TetR-Clr4*from 4xtetO-ade6+in cdc25-levels on 4xtetO-ade6+dropped by 70%within one cell cycle after the addition of AHT to these syn-chronized cultures,and no accelerated H3K9me2loss was evident during S phase,which is coin-cident with septation (21).We also released TetR-Clr4*from 4xtetO-ade6+in noncycling G 2blocked cdc25-22cells (Fig.3B).TetR-Clr4*was lost from 4xtetO-ade6within 1hour,and H3K9me declined to less than 25%of initial levels within 4hours.Thus,after release of the initiating methyltrans-ferase,rather than being passively diluted through chromatin replication,H3K9methylation must be removed by an active process.Known and putative histone demethylases might act to remove H3K9me and thus disassemble heterochromatin from TetR-Clr4*tethering sites.We therefore tested whether mutation of genes for six JmjC domain (Epe1,Jmj1,Jmj2,Jmj4,Lid2,and Msc1)(22)or two SWIRM/amino-oxidase domain proteins (Lsd1and Lsd2)(23)allowed long-term 4xtetO-ade6+silencing after tethered TetR-Clr4*release.WT 4xtetO-ade6+TetR-Clr4*cells form red/ade6-repressed colonies on indicator plates lacking AHT,whereas white/ade6-expressing colonies appear on +AHT plates due to loss of H3K9me-dependent heterochromatin over 4xtetO-ade6+.Of the eight tested mutants,only epe1D consistently formed red-pink colonies on +AHT plates,indicating that 4xtetO-ade6+can remain repressed without bound TetR-Clr4*(Fig.4A and figs.S5and S6).Catalytically inactivating muta-tions in the Fe(II)or 2-oxyglutarate binding sites and epe1-K314A )had a similar phenotype (Fig.4A,fig.S6,and table S3).The variable silencing and colony color most likely reflects stochastic events at the 4xtetO-ade6+locus in epe1D cells in which H3K9me domains are known to expand and additional heterochromatin islands also ap-pear,potentially titrating and redistributing het-erochromatin proteins between various loci in individual cells (24–27).Maintenance of the si-lenced state in epe1D cells is not dependent on the RNAi component Ago1,as ago1D epe1D cells form red/ade6-silent colonies on +AHT plates (fig.S7A),but it does require untethered WT Clr4with an intact Clr4chromodomain and Swi6(fig.S8).This reliance on untethered,intact Clr4and Swi6is consistent with a simple read-write prop-agation mechanism (fig.S10).Silencing of 4xtetO-ade6+can be propagated through multiple cell divisions in epe1mutants (lost in 4%of cells per division),and a high pro-portion of descendant cells retain silencing of,and 30to 70%of H3K9me2on,4xtetO-ade6+after TetR-Clr4*release by AHT.In contrast,4xtetO-ade6+silencing and H3K9me2are completely lost in WT cells (Fig.4A and fig.S7,B to E).The rel-ative levels of H3K9me2and H3K9me3detected on 4xtetO-ade6+are similar in WT and epe1D cells,and surrounding genes are silenced by H3K9me2in both WT and epe1D .(fig.S9).To determine whether H3K9me on 4xtetO-ade6+in epe1D cells is maintained through meiosis in the absence of TetR-Clr4*,epe1D 4xtetO-ade6+tetR-clr4*cells (F 0)were crossed to epe1D cells devoid of both 4xtetO-ade6+and TetR-Clr4*,and then F 1epe1D 4xtetO-ade6+progeny lacking TetR-Clr4*were again crossed to epe1D cells.A high pro-portion of resulting F 2epe1D 4xtetO-ade6+pro-geny formed red-pink/ade6-repressed colonies,and H3K9me2was retained (Fig.4B and fig.S6B).Thus,epe1D allows silencing and H3K9me to persist through multiple mitotic divisions and meiosis,in the complete absence of the tethered TetR-Clr4*that initiated H3K9me-dependent het-erochromatin on 4xtetO-ade6+.Crossing of red F2epe1D 4xtetO-ade6+cells to WT epe1+cells resulted in loss of silencing (white colonies only)and H3K9me2from the 4xtetO-ade6+locus.Thus,provision of epe1+results in removal of persistent H3K9me and loss of silencing (Fig.4B).Genet-ically identical naïve epe1D 4xtetO-ade6+cells that were never exposed to the TetR-Clr4*initiator formed only white/ade6-expressing colonies,and H3K9me2was absent (Fig.4B).We conclude that the transient tethering of TetR-Clr4*adjacent to 4xtetO-ade6+allows establishment of H3K9me-dependent heterochromatin,which can be propa-gated epigenetically through mitotic cell divisions and meiosis using endogenous read-write copying mechanisms,provided that Epe1is rendered non-functional (for a model,see fig.S10).Propagation of heterochromatin on 4xtetO-ade6+in epe1mutants requires recognition of TetR-Clr4*–mediated H3K9me by the chromodomain of Clr4and also Swi6(fig.S8).Epe1associates with Swi6HP1and clearly opposes heterochromatin formation (24–28).Indeed,Epe1associates with TetR-Clr4*–mediated heterochromatin (fig.S7C).Although1343APRIL 2015•VOL 348ISSUE 6230 SCIENCERESEARCH |REPORTSsite is unusual,and histone demethylase activity has not been detected (22).However,the human PHF2JmjC domain bears a similar anomaly but phosphorylation activates its latent H3K9demeth-ylase activity (29).The analyses presented here are consistent with Epe1normally acting as an H3K9demethylase that removes H3K9methyl-ation from ectopic sites of heterochromatin for-mation.Moreover,additional heterochromatin islands and domain expansion in epe1mutants are best explained by the loss of an H3K9demeth-ylase that prevents excessive H3K9me-dependent heterochromatin formation.Epe1-dependent re-moval of H3K9me ensures regulation of centro-meric heterochromatin and makes the RNAi pathway essential for the systematic replenish-ment of H3K9me every cell cycle (30,31).Epe1itself may be regulated in response to environ-mental cues to retain or eliminate H3K9methyl-ation at specific locations (26).Indeed,Epe1levels are regulated,and this may aid the persistence of centromeric H3K9me-dependent heterochromatin (28).Thus,opposing H3K9methyltransferase and demethylase activities must be finely tuned to allow controlled heterochromatin formation and prevent its inappropriate mitotic and trans-generational inheritance.It seems counterin-tuitive for heterochromatin to carry a means of self-destruction;however,such an inbuilt safety mechanism averts the inappropriate,and poten-tially deleterious,silencing of genes by removingpropagation.REFERENCES AND NOTES1.G.Almouzni,A.V.Probst,Nucleus 2,332–338(2011).2. A.J.Bannister et al .,Nature 410,120–124(2001).chner,D.O ’Carroll,S.Rea,K.Mechtler,T.Jenuwein,Nature 410,116–120(2001).4.K.Zhang,K.Mosch,W.Fischle,S.I.Grewal,Nat.Struct.Mol.Biol.15,381–388(2008).5.T.Wang et al .,PLOS ONE 7,e52977(2012).6.S.A.Jacobs,W.Fischle,S.Khorasanizadeh,Methods Enzymol.376,131–148(2003).7.L.Aagaard et al .,EMBO J.18,1923–1938(1999).8. C.Rivera,Z.A.Gurard-Levin,G.Almouzni,A.Loyola,Biochim.Biophys.Acta 1839,1433–1439(2014).9. B.Zhu,D.Reinberg,Cell Res.21,435–441(2011).10.X.Cheng,Cold Spring Harbor Perspect.Biol.6,a018747(2014).11.N.A.Hathaway et al .,Cell 149,1447–1460(2012).12.J.Nakayama,J.C.Rice,B.D.Strahl,C.D.Allis,S.I.Grewal,Science 292,110–113(2001).13.F.E.Reyes-Turcu,S.I.Grewal,Curr.Opin.Genet.Dev.22,156–163(2012).14.S.E.Castel,R.A.Martienssen,Nat.Rev.Genet.14,100–112(2013).15.A.Buscaino et al .,EMBO J.32,1250–1264(2013).16.B.S.Wheeler,B.T.Ruderman,H.F.Willard,K.C.Scott,Genetics 190,549–557(2012).17.S.Jia,K.Noma,S.I.S.Grewal,Science 304,1971–1976(2004).18.A.Kagansky et al .,Science 324,1716–1719(2009).19.Materials and methods are available as supplementarymaterials on Science Online.20.T.Sakuno,K.Tada,Y.Watanabe,Nature 458,852–858(2009).21.S.M.Kim,J.A.Huberman,EMBO J.20,6115–6126(2001).23.Y.Shi et al .,Cell 119,941–953(2004).24.M.Zofall,S.I.S.Grewal,Mol.Cell 22,681–692(2006).25.N.Ayoub et al .,Mol.Cell.Biol.23,4356–4370(2003).26.M.Zofall et al .,Science 335,96–100(2012).27.S.C.Trewick,E.Minc,R.Antonelli,T.Urano,R.C.Allshire,EMBO J.26,4670–4682(2007).28.S.Braun et al .,Cell 144,41–54(2011).29.A.Baba et al .,Nat.Cell Biol.13,668–675(2011).30.E.S.Chen et al .,Nature 451,734–737(2008).31.A.Kloc,M.Zaratiegui,E.Nora,R.Martienssen,Curr.Biol.18,490–495(2008).ACKNOWLEDGMENTSWe thank I.Stancheva and the Allshire lab for valuable discussions;E.S.Choi for RNA-seq data;and S.Grewal,F.van Leeuwen,L.Bayne,Y.Shi,H.D.Madhani,T.Urano,and H.Watanabe for providing strains and materials.P.N.C.B.A.was supported by the Wellcome Trust 4Year PhD program in Cell Biology (grant 093852).R.C.A.is supported by a Wellcome Trust Principal Research Fellowship (grant 095021),the EC-NOE-EpiGeneSys (grant HEALTH-F4-2010-257082),and core funding to theWellcome Trust Centre for Cell Biology (grant 092076).RNA-seq data have been deposited with the National Center for Biotechnology Information Gene Expression Omnibus under accession code SRX689922.SUPPLEMENTARY MATERIALS/content/348/6230/132/suppl/DC1Materials and Methods Figs.S1to S10Tables S1to S3Reference (32)1September 2014;accepted 26January 201510.1126/science.1260638SCIENCE 3APRIL 2015•VOL 348ISSUE 6230135epe1mutants retain RESEARCH |REPORTSDOI: 10.1126/science.1260638, 132 (2015);348 Science et al.Pauline N. C. B. Audergon Restricted epigenetic inheritance of H3K9 methylationThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): October 13, 2015 (this information is current as of The following resources related to this article are available online at/content/348/6230/132.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/suppl/2015/04/01/348.6230.132.DC1.htmlcan be found at:Supporting Online Material /content/348/6230/132.full.html#related found at:can be related to this article A list of selected additional articles on the Science Web sites /content/348/6230/132.full.html#ref-list-1, 10 of which can be accessed free:cites 31 articles This article/cgi/collection/molec_biol Molecular Biologysubject collections:This article appears in the following registered trademark of AAAS.is a Science 2015 by the American Association for the Advancement of Science; all rights reserved. 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- 160 -*基金项目：国家自然科学基金项目（82260483；81502556）；云南省科技厅基础研究专项-重点项目（202301AS070015）；云南省消化内镜临床医学中心项目（2022LCZXKF-XH19）①昆明理工大学医学院　云南　昆明　650500②云南省第一人民医院通信作者：郭强SMAD3对消化道恶性肿瘤调控作用的研究进展*李西沙①　唐慧②　刘中建②　郭强② 【摘要】　消化道恶性肿瘤的发生及进展机制一直是国内外研究的热点。

SMAD3作为一种受体调节型蛋白，参与了癌症信号通路，对多数消化道恶性肿瘤的增殖、迁移及侵袭等起着重要的调控作用，与肿瘤患者的预后密切相关。

本文就SMAD3的结构功能、其在消化道恶性肿瘤中的作用机制的最新研究做一综述，以对SMAD3有更全面的了解。

【关键词】　SMAD3　消化道恶性肿瘤　癌基因 Research Progress on the Regulatory Effect of SMAD3 on Gastrointestinal Malignant Tumor/LI Xisha, TANG Hui, LIU Zhongjian, GUO Qiang. //Medical Innovation of China, 2023, 20(36): 160-164 [Abstract]　The occurrence and progression mechanism of gastrointestinal malignant tumor have been the focus of research at home and abroad. As a receptor regulated protein, SMAD3 is involved in cancer signaling pathway and plays an important regulatory role in the proliferation, migration and invasion of most gastrointestinal malignant tumor, which is closely related to the prognosis of tumor patients. In this paper, the structure and function of SMAD3 and its mechanism of action in gastrointestinal malignant tumor are reviewed, so as to have a more comprehensive understanding of SMAD3. [Key words]　SMAD3　Gastrointestinal malignant tumor　Oncogene First-author's address: School of Medicine, Kunming University of Science and Technology, Kunming 650500, China doi：10.3969/j.issn.1674-4985.2023.36.036 肿瘤的发生发展是一个复杂多步骤的生物学过程，多基因参与了肿瘤的调控，在肿瘤的调控网络中存在着一些关键基因。

2015年生物科研界的八件大事一、发现磁感应蛋白，解密第六感之谜北京大学生命科学学院的研究人员在《Nature Material》杂志发表论文,公开了一种全新的磁受体蛋白（MagR）。

该突破性进展或将揭开被称为生物“第六感”的磁觉之谜，并推动整个生物磁感受能力研究领域的发展。

在自然界，许多动物物种都有感知地球磁场的能力。

它们能感知磁场的方向、强度或者倾斜度，并且把此类信息作为导航线索，但科研人员对这些的生物学机制并不清楚。

研究团队通过用假定的生物标准筛选果蝇基因组，发现了一种像聚合物的蛋白质——MagR。

这种蛋白质会和光敏隐花色素蛋白的组成部分结合在一起，自发地和外部磁场对齐。

研究人员通过生物化学和生物物理的方法发现，缺少光敏隐花色素蛋白的果蝇并不具有对磁场感应的能力。

这表明光敏隐花色素蛋白是使果蝇产生磁感应能力的必要条件，然而理论上只有光敏印花色素蛋白又不能形成“指南针”的作用。

因此研究人员认为是磁感应蛋白与光敏印花色素蛋白相结合才使动物对地磁场具有感知能力。

生物物理学和物理学实验证明，MagR蛋白复合物具有很明显的内禀磁矩，能通过磁场在实验室富集和纯化得到。

课题组不仅从物理性质上测量了该蛋白在溶液状态下的磁性特征，还通过电镜观察到MagR蛋白质复合物能感应到微弱的地球磁场，并沿着地球磁场排列。

MagR介导动物对磁场的感知有可能是构成动物迁徙和生物导航的基础。

发现这样如同指南针一般的蛋白质复合体，使得磁感应蛋白在未来利用磁场调控生物过程方面拥有广阔的应用前景。

二、细胞也会产生“错觉”人的大脑会产生感知偏差，从而产生错觉。

现在加州大学旧金山分校的研究团队发现，单细胞的酵母也会被精心设计的错觉迷惑，并因此而死亡。

研究人员发现，酵母会将特定频率的压力模式（盐浓度振荡）看成大规模持续性的压力增涨，结果做出过度反应导致自身死亡。

研究成果发表在《Science》杂志上。

正常情况下，酵母细胞内的感知分子检测盐浓度变化，并指挥细胞做出相应的应答，生产一种保护性的化合物。

异氟醚通过BMP4/Smad信号通路对自发性高血压大鼠脑损伤的作用机制研究杨雪梅1,杨康明1,李娜2摘要目的:探究异氟醚通过调节骨发生形态蛋白4(BMP4)及其下游Smad蛋白对自发性高血压大鼠(SHR)脑损伤的作用机制㊂方法:采用随机数字表法将48只SHR分为模型组㊁异氟醚组㊁阳性药物组㊁异氟醚+LDN193189组,健康大鼠作为健康对照组,每组12只㊂各组大鼠分别放入氧气箱中,健康对照组㊁模型组㊁阳性药物组大鼠通入特殊气体(30%O2㊁70%N2)1h,异氟醚组㊁异氟醚+ LDN193189组通入混杂2%异氟醚的特殊气体1h,通过麻醉气体检测仪检测异氟醚含量,确保异氟醚浓度始终保持在2%㊂通气结束后,异氟醚+LDN193189组大鼠腹腔内注射BMP4/Smad通路抑制剂溶液20μL(LDN193189,5mg/kg),阳性药物组大鼠腹腔内注射缬沙坦溶液20μL(10mg/kg),健康对照组㊁模型组㊁异氟醚组大鼠腹腔内注射等量的生理盐水㊂氧气箱通气实验及注射每日1次,持续10d㊂无创血压检测仪检测尾动脉收缩压变化;神经学行为评分评价行为功能;脑含水量检测脑水肿程度;原位末端转移酶标记法(TUNEL)染色和尼氏染色观察海马组织病理学变化及神经元损伤情况;荧光定量聚合酶链式反应(PCR)及蛋白免疫印迹(Western Blot)法检测海马组织BMP4㊁Smad2mRNA及蛋白表达水平㊂结果:与模型组比较,异氟醚组㊁阳性药物组㊁异氟醚+ LDN193189组大鼠海马神经细胞凋亡减少,尼氏小体增多,血压㊁神经学评分㊁脑组织含水量显著降低,BMP4㊁Smad2mRNA及蛋白表达量显著升高(P<0.05);与异氟醚组比较,异氟醚+LDN193189组大鼠神经细胞凋亡增多,尼氏小体急剧减少,血压㊁神经学评分㊁脑组织含水量显著升高,BMP4㊁Smad2mRNA及蛋白表达量显著降低(P<0.05);异氟醚组与阳性药物组各项数据差异均无统计学意义(P>0.05)㊂结论:异氟醚可通过促进BMP4/Smad信号通路减轻SHR脑损伤㊂关键词自发性高血压;脑损伤;异氟醚;骨发生形态蛋白4;Smad蛋白d o i:10.12102/j.i s s n.1672-1349.2024.04.010Effect of Isoflurane on Cerebral Injury in Spontaneously Hypertensive Rats through BMP4/Smad Signaling PathwayYANG Xuemei,YANG Kangming,LI NaGanzi Tibetan Autonomous Prefecture People's Hospital,Ganzi626000,Sichuan,ChinaCorresponding Author LI Na,E-mail:**************Abstract Objective:To explore the effect of isoflurane on brain injury in spontaneously hypertensive rats(SHR)by regulating bone morphogenetic protein4(BMP4)and its downstream Smad protein.Methods:Forty-eight SHR rats were randomly divided into model group,isoflurane group,positive drug group,isoflurane+LDN193189group,and healthy rats were used as healthy control group,with12 rats in each group.Rats in each group were placed into oxygen tanks,healthy control group,model group and positive drug group were injected with special gas(30%O2,70%N2)for1h,isoflurane group and isoflurane+LDN193189group were injected with special gas mixed with2%isoflurane for1h,and isoflurane content was detected by anesthesia gas detector.Ensure isoflurane concentration was maintained at2%at all times.After ventilation,rats in isoflurane+LDN193189group were intraperitoneally injected with BMP4/Smad pathway inhibitor solution of20μL(LDN193189,5mg/kg),the positive drug group was intraperitoneally injected with20μL(10mg/kg)of valsartan solution,and the healthy control group,model group and isoflurane group were intraperitoneally injected with the same volume of normal saline water.Oxygen tank ventilation test and injection were once a day for10days.The changes of tail artery systolic blood pressure were detected by non-invasive blood pressure detector.Neurological behavior score was used to evaluate behavioral function.Cerebral water content was used to measured the degree of cerebral edema.The histopathological changes and neuronal injury of hippocampus were detected by terminal deoxyribonucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL)and Nysch staining.Fluorescence quantitative polymerase chain reaction(PCR)and Western Blot were used to detect the mRNA and protein expression levels of BMP4and Smad2mRNA and protein in hippocampus.Results:Compared with the model group,the apoptosis of hippocampal nerve cells in isoflurane group,positive drug group and isoflurane+LDN193189group decreased, the number of nissellite bodies increased,the blood pressure,neurological score,and cerebral tissue water content decreased significantly,and the mRNA and protein expressions of BMP4and Smad2increased significantly(P<0.05).Compared with the isoflurane group,the neuronal apoptosis of rats in isoflurane+LDN193189group increased,the nissellite bodies decreased sharply,the blood pressure,neurological score,and cerebral tissue water content significantly increased,and the mRNA and protein expressions of BMP4and Smad2significantly decreased(P<0.05).There was no significant difference between isoflurane group and positive drug group(P>0.05).Conclusion:Isoflurane can reduce SHR cerebral damage by promoting BMP4/Smad signaling pathway.Keywords spontaneous hypertension;brain damage;isoflurane;bone morphogenetic protein4;Smad protein作者单位 1.甘孜藏族自治州人民医院(四川甘孜626000);2.海南省人民医院(海口572000)通讯作者李娜,E-mail:**************引用信息杨雪梅,杨康明,李娜.异氟醚通过BMP4/Smad信号通路对自发性高血压大鼠脑损伤的作用机制研究[J].中西医结合心脑血管病杂志, 2024,22(4):640-645.高血压为我国常见的慢性疾病之一,随着人口老龄化的加剧,高血压疾病发生率逐渐增高,且由于饮食作息等因素,近年来高血压病人趋于年轻化[1-3]㊂目前,针对高血压疾病的治疗药物较多,且多为西药,尽管具有较好的疗效,但易出现一定的副作用[4-5]㊂高血压发病原因多样,受多条通路调控[6]㊂骨发生形态蛋白4(bone morphogenetic protein4,BMP4)具有诱导骨形成的作用,不仅大量存在于骨组织中,在中枢神经系统中也高表达,调节神经元生长,在神经受损后加速神经细胞的生长与分化,从而缓解脑损伤,BMP4通常与其下游蛋白Smad结合,两者共同发挥作用[7-8]㊂异氟醚是一种具有乙醚样气味的特殊有机物,作为常见的吸入类麻醉药物,常用于手术的全身麻醉,具有良好的安全性[9-10]㊂研究发现,异氟醚对于脑缺血引起的脑损伤具有良好的治疗效果,已被应用于治疗缺血缺氧性脑损伤病人的神经损伤,对脑神经有良好的保护作用[11]㊂异氟醚可通过调控BMP4/Smad信号通路相关基因的表达缓解缺血再灌注损伤大鼠的脑损伤[12]㊂本研究以自发性高血压大鼠(spontaneously hypertensive rats,SHR)为实验模型,探究异氟醚是否通过调控BMP4/Smad信号通路对SHR脑损伤产生影响㊂1材料与方法1.1主要试剂与仪器异氟醚(货号080906)购于美国艾伯维公司,缬沙坦胶囊(国药准字H20040217,用生理盐水配成溶液)购于北京诺华制药有限公司,BMP4/Smad通路抑制剂(LDN193189,货号HY-12071)购于MedChemExpress 公司,原位末端转移酶标记法(TUNEL)染色试剂盒(货号FA201-01)购于北京全式金生物技术有限公司,尼氏染色试剂盒(货号G1432)购于北京索莱宝科技有限公司,3-磷酸甘油醛脱氢酶(GAPDH)抗体(货号ab9485)㊁BMP4抗体(货号ab124715)㊁Smad2抗体(货号ab280888)购于Abcam公司,羊抗兔二抗(货号7074S)购于Cell Signaling Technology公司㊂荧光定量聚合酶链式反应(PCR)仪(7500,ABI公司生产),小动物无创血压检测仪(panlab NIBP,深圳市瑞沃德生命科技有限公司生产),凝胶成像系统(HE-120,上海生工生物有限公司生产)㊂1.2动物分组及干预由河北省实验动物中心购得8周龄雄性Wistar SHR48只㊁8周龄雄性健康Wistar大鼠12只,许可证号SCXK(冀)2018-004,体质量(250ʃ35)g㊂通过神经学评分[13]评估大鼠行为功能,SHR评分均ȡ3分,健康大鼠评分均为0分,模型有效,其中,神经学评分0分为无抽搐反应,正常行走;1分为出现面部痉挛,伴肌痉挛;2分为间歇性抽搐;3分为伴剧烈强直性痉挛抽搐;4分为全身阵挛抽搐伴站立不稳;5分为难以站立,抽搐时间延长㊂采用随机数字表法将SHR分为模型组㊁异氟醚组㊁阳性药物组㊁异氟醚+LDN193189组,健康大鼠作为健康对照组,每组12只㊂各组大鼠分别放入氧气箱中,健康对照组㊁模型组㊁阳性药物组大鼠通入特殊气体(30%O2㊁70%N2)1h,异氟醚组㊁异氟醚+LDN193189组通入混杂2%异氟醚的特殊气体1h,通过麻醉气体检测仪检测异氟醚含量,确保异氟醚浓度始终保持在2%㊂通气结束后,异氟醚+ LDN193189组大鼠腹腔内注射BMP4/Smad通路抑制剂溶液20μL(LDN193189,5mg/kg),阳性药物组大鼠腹腔内注射缬沙坦溶液20μL(10mg/kg),健康对照组㊁模型组㊁异氟醚组大鼠腹腔内注射等量的生理盐水㊂氧气箱通气实验及注射每日1次,持续10d㊂末次干预24h后,通过神经学评分评估大鼠行为功能㊂1.3大鼠血压监测各组大鼠在首次氧气箱通气实验前及末次干预24h后,用恒温水(40ħ)持续冲洗大鼠尾部,使大鼠尾部松软并尾动脉扩张,毛巾擦干尾部后,在大鼠尾部套入小动物无创血压检测仪尾套,待血压测定仪界面出现稳定脉搏信号后,开始检测尾动脉收缩压,记录统计结果㊂1.4大鼠脑组织含水量检测末次检测血压后,处死各组大鼠,解剖获取大鼠脑组织,取出海马组织,部分组织置于4%多聚甲醛中固定备用,部分组织置液氮处理后研磨成粉,冻存备用㊂剩余脑组织称重,记作湿重,而后将脑组织放入烘干箱中,持续烘干24h,确保烘干水分,而后取出称量,记作干重㊂通过脑组织含水量计算公式计算脑组织含水量= (湿重-干重)/湿重ˑ100%㊂1.5TUNEL染色及尼氏染色检测神经损伤情况取1.4中在多聚甲醛中固定24h后的海马组织,不同浓度乙醇梯度脱水,二甲苯染色透明,石蜡包埋后切片(厚度5μm),脱蜡至水,分别按照TUNEL染色试剂盒和尼氏染色试剂盒的制造商说明对切片进行处理,最后在光镜下观察染色结果并拍照㊂1.6荧光定量PCR检测海马组织BMP4㊁Smad2mRNA水平取1.4中冻存备用的海马组织粉末,使用Trizol试剂提取海马组织总RNA,加入焦碳酸二乙酯(DEPC)水溶解,检测提取的RNA浓度,定量后反转录为cDNA㊂以cDNA为模板在荧光定量PCR仪上检测BMP4㊁Smad2mRNA水平㊂以GAPDH为内参,采用2-әәCt法计算BMP4㊁Smad2的相对表达水平㊂mRNA引物序列如下:GAPDH正向引物为5'-AGTTCAACGGCACAGTCAAG-3',反向引物为5'-TACTCAGCACCAGCATCACC-3';BMP4正向引物为5'-AGAAATGGTGCCTGGAC ACCTCAT-3',反向引物为5'-TGGTCCCGGTTGTACAGTCCTAAT-3'; Smad2正向引物为5'-CCACT ACCAGAGGGTGGAGA-3',反向引物为5'-CCTGCTGGGAAATTTG TGTT-3'㊂1.7蛋白免疫印迹(Western Blot)法检测海马组织中BMP4㊁Smad2蛋白表达取出剩余的海马组织粉末,加入蛋白裂解液充分震荡,溶解蛋白后加入蛋白提取液,再次震荡,静置片刻,4ħ下10000r/min离心10min,收集上层清液进行蛋白定量㊂将定量后的蛋白经电泳㊁转膜处理,然后用5%脱脂奶粉封闭2h,加入一抗(GAPDH抗体㊁BMP4抗体㊁Smad2抗体,1ʒ1000),4ħ孵育过夜㊂倒出封闭液,用TBST缓冲液清洗3次,每次3min㊂加入羊抗兔二抗(1ʒ1500),常温孵育40min,TBST 清洗3次,每次3min㊂将发光液混合后倒于膜上,避光染色1min后吸净多余液体,置于光密度扫描系统进行条带灰度分析㊂1.8统计学处理采用SPSS20.0软件进行数据分析㊂符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,组间比较采用单因素方差分析(one-way ANOVA),进一步两两比较采用SNK-q检验㊂以P<0.05为差异有统计学意义㊂2结果2.1异氟醚对SHR神经学评分及血压的影响与健康对照组比较,模型组大鼠干预前后神经学评分及血压显著升高(P<0.05);与本组干预前比较,健康对照组㊁模型组大鼠干预后神经学评分及血压差异无统计学意义(P>0.05),阳性药物组㊁异氟醚组㊁异氟醚+LDN193189组干预后神经学评分及血压显著降低(P<0.05)㊂与模型组干预后比较,阳性药物组㊁异氟醚组㊁异氟醚+LDN193189组大鼠干预后神经学评分及血压显著降低(P<0.05);与异氟醚组干预后比较,异氟醚+LDN193189组大鼠干预后神经学评分及血压显著升高(P<0.05),干预前神经学评分及血压差异均无统计学意义(P>0.05);异氟醚组与阳性药物组比较,大鼠干预前后神经学评分及血压差异均无统计学意义(P>0.05)㊂详见表1㊂表1各组大鼠神经学评分及血压比较(xʃs)组别只数时间神经学评分(分)血压(mmHg)健康对照组12干预前0.00131.86ʃ8.71干预后0.00132.81ʃ7.32模型组12干预前 3.21ʃ0.13②184.84ʃ7.53②干预后 3.24ʃ0.15②182.88ʃ8.89②阳性药物组12干预前 3.26ʃ0.12②187.83ʃ6.39②干预后 1.17ʃ0.18①②③146.25ʃ7.36①②③异氟醚组12干预前 3.19ʃ0.21②183.36ʃ7.41②干预后 1.21ʃ0.21①②③143.71ʃ8.71①②③异氟醚+LDN193189组12干预前 3.18ʃ0.19②187.29ʃ6.81②干预后 2.31ʃ0.25①②③④161.34ʃ6.31①②③④注:与本组干预前比较,①P<0.05;与健康对照组同时间比较,②P<0.05;与模型组干预后比较,③P<0.05;与异氟醚组干预后比较,④P<0.05㊂2.2异氟醚对SHR脑水肿的影响与健康对照组比较,模型组大鼠脑组织含水量显著升高(P<0.05);与模型组比较,阳性药物组㊁异氟醚组㊁异氟醚+LDN193189组大鼠脑组织含水量显著降低(P<0.05);与异氟醚组比较,异氟醚+ LDN193189组大鼠脑组织含水量显著升高(P<0.05);异氟醚组与阳性药物组比较,大鼠脑组织含水量差异无统计学意义(P>0.05)㊂详见表2㊂表2各组大鼠脑组织含水量比较(xʃs)单位:%组别只数脑组织含水量健康对照组1273.72ʃ4.15模型组1289.47ʃ5.21①阳性药物组1279.26ʃ3.01①②异氟醚组1280.19ʃ2.81①②异氟醚+LDN193189组1284.58ʃ3.19①②③注:与健康对照组比较,①P<0.05;与模型组比较,②P<0.05;与异氟醚组比较,③P<0.05㊂2.3异氟醚对SHR神经损伤的影响TUNEL染色及尼氏染色显示,健康对照组大鼠海马组织中神经元细胞排列紧密,有大量均匀分布的蓝色颗粒状尼氏小体,未出现凋亡神经元㊂与健康对照组比较,模型组大鼠海马组织中神经元细胞排列松散,尼氏小体减少,观察到大量神经元细胞凋亡;与模型组比较,阳性药物组㊁异氟醚组大鼠海马组织中神经元细胞排列较为密集,尼氏小体逐渐增多,神经元细胞凋亡减少;与异氟醚组比较,异氟醚+LDN193189组尼氏小体较少,神经细胞凋亡增多,且组织中神经元细胞排列分散㊂详见图1㊁图2㊂图1各组大鼠海马组织尼氏染色后神经元细胞变化(ˑ400)图2各组大鼠海马组织TUNEL染色后神经元细胞变化(ˑ400)2.4异氟醚对SHR海马组织BMP4㊁Smad2mRNA 表达的影响与健康对照组比较,模型组大鼠海马组织BMP4㊁Smad2mRNA表达显著降低(P<0.05);与模型组比较,阳性药物组㊁异氟醚组㊁异氟醚+LDN193189组大鼠海马组织BMP4㊁Smad2mRNA表达显著升高(P<0.05);与异氟醚组比较,异氟醚+LDN193189组大鼠海马组织BMP4㊁Smad2mRNA表达显著降低(P< 0.05);阳性药物组与异氟醚组比较,大鼠海马组织BMP4㊁Smad2mRNA表达差异无统计学意义(P> 0.05)㊂详见表3㊂表3各组大鼠海马组织BMP4㊁Smad2mRNA表达比较(xʃs)组别只数BMP4mRNA Smad2mRNA 健康对照组12 1.06ʃ0.08 1.08ʃ0.12模型组120.27ʃ0.09①0.31ʃ0.07①阳性药物组120.81ʃ0.07②0.76ʃ0.06②异氟醚组120.78ʃ0.12②0.82ʃ0.11②异氟醚+LDN193189组120.51ʃ0.12②③0.47ʃ0.06②③注:模型组与健康对照组比较,①P<0.05;与模型组比较,②P<0.05;与异氟醚组比较,③P<0.05㊂2.5异氟醚对SHR海马组织BMP4㊁Smad2蛋白表达的影响与健康对照组比较,模型组大鼠海马组织BMP4㊁Smad2蛋白表达量显著降低(P<0.05);与模型组比较,阳性药物组㊁异氟醚组㊁异氟醚+LDN193189组大鼠海马组织BMP4㊁Smad2蛋白表达量显著升高(P< 0.05);与异氟醚组比较,异氟醚+LDN193189组大鼠海马组织BMP4㊁Smad2蛋白表达量显著降低(P< 0.05);阳性药物组与异氟醚组比较,大鼠海马组织BMP4㊁Smad2蛋白表达量差异无统计学意义(P>0.05)㊂详见图3㊁表4㊂图3各组SHR大鼠海马组织BMP4㊁Smad2蛋白表达条带图(A为健康对照组;B为模型组;C为阳性药物组; D为异氟醚组;E为异氟醚+LDN193189组)表4各组SHR大鼠海马组织BMP4㊁Smad2蛋白表达比较(xʃs)组别只数BMP4蛋白Smad2蛋白健康对照组12 3.51ʃ0.28 2.58ʃ0.32模型组120.52ʃ0.26①0.35ʃ0.16①阳性药物组12 2.81ʃ0.37② 1.95ʃ0.14②异氟醚组12 2.78ʃ0.32② 1.89ʃ0.21②异氟醚+LDN193189组120.89ʃ0.22②③0.76ʃ0.24②③注:模型组与健康对照组比较,①P<0.05;与模型组比较,②P<0.05;与异氟醚组比较,③P<0.05㊂3讨论高血压作为我国发病率较高的疾病,严重危害人类健康,长期的高血压会导致脑动脉粥样硬化,引起脑缺血,造成脑组织水肿及神经功能障碍[14-15]㊂异氟醚常作为麻醉用药用于临床试验,已证实可参与缓解脑损伤,且在临床应用,异氟醚预处理可帮助体外循环手术病人缓解脑损伤,改善认知功能[16]㊂同时对重度脑损伤病人脑部具有保护作用,可通过降低脑氧代谢率,缓解神经损伤[17]㊂异氟醚还可与其他通用麻醉类药物如丙泊酚等共同使用,麻醉同时缓解脑部损伤[18]㊂缬沙坦是治疗高血压及保护脑损伤的常用药物,故本研究选择缬沙坦作为阳性对照[19]㊂本研究结果显示,模型组大鼠神经学评分及血压显著升高,说明实验所用SHR确有高血压及高血压引起的脑组织损伤,异氟醚处理及阳性药物处理后,大鼠神经学评分及血压均显著降低,表明异氟醚具有与常规药物相同的治疗效果,可在降低高血压的同时,减轻脑损伤引起的行为功能障碍㊂研究显示,海马组织作为中枢神经系统中重要的记忆组织,含有大量神经元,是判断脑损伤的重要组织;脑组织含水量是评判脑水肿的重要标志[20-21]㊂本研究结果显示,SHR海马组织尼氏小体大量减少,大量神经元细胞凋亡,脑组织含水量显著升高;阳性药物缬沙坦及异氟醚处理可增加尼氏小体数量,抑制神经元细胞凋亡,降低脑组织含水量,提示异氟醚可以缓解SHR海马组织神经元损伤,减轻脑水肿,维持脑组织结构正常,但具体的作用机制尚未明确㊂研究显示,BMP4在中枢神经系统中发挥重要的保护作用,在脑缺血引起的脑损伤过程中参与保护神经细胞,从而缓解脑损伤带来的神经损害,BMP4与下游蛋白Smad结合,共同维护神经组织,缓解脑水肿等多种脑组织损害[22]㊂同时有研究指出,异氟醚可通过调控BMP4/Smad通路相关基因表达影响脑缺血再灌注损伤大鼠的症状[23]㊂故推测异氟醚可能通过调控BMP4/Smad信号通路参与对SHR脑损伤的保护作用㊂本研究结果显示,SHR海马组织中BMP4㊁Smad2 mRNA和蛋白表达显著降低,而异氟醚处理可提高大鼠海马组织BMP4㊁Smad2mRNA和蛋白表达,说明BMP4/Smad信号通路参与SHR发生,而异氟醚很可能通过调控BMP4/Smad信号通路对SHR发挥作用㊂进一步研究显示,BMP4/Smad通路抑制剂可逆转异氟醚对SHR脑损伤的保护作用,再次验证了异氟醚通过促进BMP4/Smad信号通路参与缓解SHR脑损伤㊂综上所述,异氟醚可通过促进BMP4/Smad信号通路降低SHR血压,并减轻大鼠脑神经元凋亡及脑水肿,缓解脑组织损伤,减轻大鼠行为功能障碍,从而治疗和改善高血压及其并发症㊂异氟醚治疗高血压具有较好的疗效,且安全易操作,有很大的应用空间,但异氟醚是否还通过调节其他通路间接参与调控高血压,仍有待进一步的实验验证㊂参考文献:[1]LÜSCHER T F.Imbalance of endothelium-derived relaxing andcontracting factors:a new concept in hypertension?[J].American Journal of Hypertension,1990,3(4):317-330. [2]HALBACH M,GROTHAUS D,HOFFMANN F,et al.Baroreflexactivation therapy reduces frequency and duration ofhypertension-related hospitalizations in patients with resistanthypertension[J].Clinical Autonomic Research,2020,30(6):541-548.[3]WANG J,XU Y N,TANG L,et al.Correlation between serum totalbilirubin levels and blood pressure variability in patients withhypertension[J].Am J Hypertens,2021,34(9):1009-1010. [4]罗琼,王晓华.拉贝洛尔联合酚妥拉明治疗妊娠期高血压疾病患者的疗效及对胎盘血管内皮生长因子的影响[J].中国妇幼保健,2020,35(3):415-418.[5]WU J J,ZENG T X,LIANG J R,et al.Effects of differentacupuncture manipulations on protein expression in the parietalcortex of spontaneously hypertensive rats[J].Journal ofTraditional Chinese Medical Sciences,2021,8(3):257-264. [6]张琦,王冬冬,刘艺丹,等.甘肃省成年人高血压患病状况及影响因素研究[J].中国全科医学,2019,22(10):1197-1202.[7]彭韬,苗刚勇,谭志强,等.BMP4促进诱导多能干细胞向感觉神经元分化[J].海南医学,2019,30(18):2313-2317.[8]THOMPSON A,BERRY M,LOGAN A,et al.Activation of the BMP4/Smad1pathway promotes retinal ganglion cell survival and axonregeneration[J].Investigative Ophthalmology&Visual Science,2019,60(5):1748-1759.[9]ANAGHA M,KAMATH M R.Influence of bispectral indexmonitoring on isoflurane minimum alveolar concentration in offpump cardiac surgeries[J].Journal of Evolution of Medical andDental Sciences,2020,9(13):1058-1064.[10]TANG X L,ZHANG X,LI S Y,et al.NR2B receptor-and calpain-mediated KCC2cleavage resulted in cognitive deficiencyexposure to isoflurane[J].Neurotoxicology,2020,76:75-83.[11]杨玉琪,王胜,司军强,等.异氟烷后处理减轻大鼠局灶性脑缺血再灌注损伤时TGF-β3/Smad3信号通路与神经元凋亡的关系[J].中华麻醉学杂志,2020,40(4):416-420.[12]袁敏.AQP4及BMP4/Smad l/5/8通路在异氟醚后处理大鼠脑缺血再灌注损伤中的作用[D].石河子:石河子大学,2018. [13]ESSAWY A E,EL-SAYED S A,TOUSSON E,et al.Anti-kindlingeffect of Ginkgo biloba leaf extract and L-carnitine in thepentylenetetrazol model of epilepsy[J].Environ Sci Pollut ResInt,2022,29(32):48573-48587.[14]杜厚伟,陈超,林菲菲,等.大脑中动脉狭窄/闭塞的高血压脑梗死患者血压成分与基底节血管周围间隙的相关性[J].中华高血压杂志,2019,27(12):1189-1193.[15]徐天阳,徐挺立.高压氧治疗对高血压脑出血患者神经功能恢复及脑水肿的影响分析[J].心脑血管病防治,2019,19(3):267-269.[16]王欣,章放香.异氟醚预处理对体外循环心脏瓣膜置换术患者脑损伤的影响[J].中华麻醉学杂志,2018,38(1):88-91. [17]孙贺.丙泊酚或异氟醚对重度脑损伤患者Barthel指数的影响比较[J].国际医药卫生导报,2017,23(3):395-397.[18]魏静,蔡德学.丙泊酚与异氟醚麻醉对急性颅脑外伤患者脑保护作用比较[J].世界最新医学信息文摘,2017,17(26):132-133. [19]LOU-MEDA R,STILLER B,ANTONIO Z L,et al.Long-term safetyand tolerability of valsartan in children aged6to17years withhypertension[J].Pediatric Nephrology,2019,34(3):495-506. [20]JI M,NIU S Q,MI H Y,et al.Antidepressant functions of Jie Yu ChuFan capsule in promoting hippocampal nerve cell neurogenesisin a mouse model of chronic unpredictable mild stress[J].Annalsof Translational Medicine,2020,8(16):1020.[21]汪棋笙,丁华强,廖帅,等.丙戊酸钠对创伤性颅脑损伤大鼠脑组织中金属基质蛋白-9及水通道蛋白4表达的影响[J].医学研究生学报,2019,32(8):809-814.[22]龙婷婷,谢明,周璐,等.Noggin蛋白对小鼠脑缺血再灌注损伤后学习和记忆能力与齿状回结构的影响[J].山东大学学报(医学版),2020,58(7):15-23.[23]孟轶男,安洋,王守田.异氟醚后处理通过BMP4/Smad信号通路调节脑缺血再灌注损伤大鼠AQP4表达[J].解剖学研究,2018,40(5):398-402.(收稿日期:2021-11-17)(本文编辑邹丽)。

人眼Tenon's囊成纤维细胞原代培养的研究陈珺;李宁;廖荣丰【摘要】目的探讨体外培养原代人眼Tenon's囊成纤维细胞的方法及其生长特性,为抗纤维化研究提供靶细胞模型.方法取翼状胬肉及斜视手术患者的Tenon's囊组织,用组织块法培养原代成纤维细胞.用免疫荧光方法鉴定细胞.对细胞进行传代、冻存和复苏的观察.对复苏后的细胞行MTT法检测其活力并绘制生长曲线.结果入眼Tenon's囊成纤维细胞可以在体外用组织块方法培养出来,呈典型的长梭形.免疫荧光鉴定细胞波形蛋白染色阳性.细胞多次传代后依然生长迅速,3d即可长满瓶底.复苏后细胞2～6d处生长对数期,增殖能力良好.结论人眼Tenon's囊成纤维细胞体外生长状态良好,可以液氮冻存,复苏后细胞活力较强,可以用于抗纤维化增生的基础研究.【期刊名称】《临床眼科杂志》【年(卷),期】2016(024)001【总页数】4页(P1-4)【关键词】人Tenon囊;成纤维细胞;细胞培养技术【作者】陈珺;李宁;廖荣丰【作者单位】230022 合肥,安徽医科大学第一附属医院眼科;230022 合肥,安徽医科大学第一附属医院眼科;230022 合肥,安徽医科大学第一附属医院眼科【正文语种】中文纤维增生性疾病在多个医学领域(包括眼部、皮肤、整形和肿瘤等)中尚属治疗难点，对其发生发展和机制还有待进一步研究[1，2]。

瘢痕化的形成是其共同的表现，瘢痕化的产生主要是与多种细胞因子的产生、细胞及细胞外基质活动有关的生物学过程[3-7]。

其中人眼Tenon's囊成纤维细胞(human Tenon's capsule fibroblasts,HTFs)的过度增殖是纤维瘢痕化的主要原因，所以探索简单快速地培养出大量HTFs，观察其生长特性，研究细胞经过数次传代、液氮冻存及复苏后的活力如何，为抗瘢痕化的研究提供理想细胞模型。

一、取材本实验已经安徽医科大学第一附属医院伦理委员会审核批准，人眼Tenon's囊组织均取材于本院斜视及翼状胬肉手术患者，患者均签署知情同意书，无既往眼部手术史，术中打开结膜囊暴露Tenon's组织，取材约5 mm×5 mm大小并放入含DMEM培养液的无菌离心管中，装入无菌冰盒带回实验室。

October 2018Corresponding author(s):Sinem K. Saka, Yu Wang, Peng YinLast updated by author(s):June 05, 2019Reporting SummaryNature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist .StatisticsFor all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.The exact sample size (n ) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedlyThe statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)For null hypothesis testing, the test statistic (e.g. F , t , r ) with confidence intervals, effect sizes, degrees of freedom and P value notedGive P values as exact values whenever suitable.For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settingsFor hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomesEstimates of effect sizes (e.g. Cohen's d , Pearson's r ), indicating how they were calculatedOur web collection on statistics for biologists contains articles on many of the points above.Software and codePolicy information about availability of computer codeData collection Commercial softwares licensed by microscopy companies were utilized: Zeiss Zen 2012 (for LSM 710), Leica LAS AF (for Leica SP5), ZeissZen 2.3 Pro Blue edition (for LZeiss Axio Observer Z1), Olympus VS-ASW (for Olympus VS120), PerkinElmer Phenochart (version 1.0.2) .Data analysis Open-source Python (3.6.5), TensorFlow (1.12.0), and Deep Learning packages have been utilized for machine learning-based nucleiidentification (the algorithm and code is available at https:///HMS-IDAC/UNet). We used Matlab (2017b) for watershed-based nuclear segmentation using the identified nuclear contours. Python 3.6 was used for the FWHM calculations, as well as plotting ofhistograms. We used MATLAB and the Image Processing Toolbox R2016a (The MathWorks, Inc., Natick, Massachusetts, United States)for quantifications in mouse retina sections and for Supplementary Fig. 4. We utilized Cell Profiler 3.1.5 for the quantifications of signalamplification in FFPE samples in Figure 2 and 3. FIJI (version 2.0.0-rc-69/1.52n) was utilized for ROI selections and format conversions.HMS OMERO (version 5.4.6.21) was used for viewing images and assembling figure panels.For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). 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The MATLAB code for nuclear segmentation isOctober 2018available on: https:///HMS-IDAC/SABERProbMapSegmentation .Field-specific reportingPlease select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.Life sciencesBehavioural & social sciences Ecological, evolutionary & environmental sciencesFor a reference copy of the document with all sections, see /documents/nr-reporting-summary-flat.pdfLife sciences study design All studies must disclose on these points even when the disclosure is negative.Sample size Each FFPE experiment batch were performed on consecutive sections from the same source, each containing over 600,000 cells. Due to largenumber of single cells with tens of distinct germinal center morphologies being present in each section, ROIs from different parts of a wholesection was used for quantification of signal improvement for each condition (consecutive sections were used for all the conditions of onequantification experiment). Number of ROIs are noted in the respective figure legends. For quantifications in retina samples, due toconserved staining morphology and low sample-to-sample variability n = 6 z-stacks were acquired from at least 2 retina sections. ForSupplementary Fig. 4, minimum 5 z-stacks were acquired for each condition to collect images of 18-45 cells. Number of cells are reported in the graphs.Data exclusions Parts of the FFPE tissue sections were excluded from analysis due to automated imaging related aberrations (out-of-focus areas) or tissuepreparation aberrations (folding of the thin sections at the edges, or uneven thickness at the edge areas). For FWHM calculations inSupplementary Fig. 2, ROIs that yield lineplots with more than one automatically detected peak were discarded to avoid deviations due tomultiple peaks. For Supplementary Fig. 4 cells in the samples were excluded when an external bright fluorescent particle (dust speck, dye aggregate etc.) coincided with the nuclei (as confirmed by manual inspection of the images). The exclusion criteria were pre-established.Replication Each FFPE experiment batch were performed on consecutive sections from the same source, each containing over 600,000 cells. Forevaluation and quantification of our method, multiple biological replicates were not accumulated in order to avoid the error that would beintroduced by the natural biological and preparation variation, and to avoid unnecessary use of human tissue material. In the case of themouse retina quantifications a minimum of two distinct retinal sections were imaged, and each experiment was performed at least twice. ForSupplementary Fig. 4 dataset, 16 different conditions were prepared and each were imaged multiple times (before linear, after linear, beforebranch, after branch). Although the data was not pooled together for the statistics reported in the figure, low cell-to-cell variability was observed and high consistency was seen across the samples for comparable conditions, suggesting low sample to sample variability.Randomization Randomization was not necessary for this study.Blinding Blinding was not possible as experimental conditions were mostly evident from the image data.Reporting for specific materials, systems and methodsWe require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.AntibodiesAntibodies used The full list is also available in Supplementary Information, Supplementary Table 4.Ki-67 Cell Signaling #9129, clone: D3B5 (formulated in PBS, Lot: 2), diluted 1:100-1:250 after conjugationCD8a Cell Signaling #85336 clone: D8A8Y (formulated in PBS, Lot: 4) diluted 1:150 after conjugationPD-1 Cell Signaling #43248, clone: EH33 (formulated in PBS, Lot: 2), diluted 1:150 after conjugationIgA Jackson ImmunoResearch #109-005-011 (Lot: 134868), diluted 1:150 after conjugationCD3e Cell Signaling #85061 clone: D7A6E(TM) XP(R) (formulated in PBS, Lot:2), diluted 1:150 after conjugationIgM Jackson ImmunoResearch #709-006-073 (Lot: 133627), diluted 1:150 after conjugationLamin B Santa Cruz sc-6216 clone:C-20, (Lot: E1115), diluted 1:100Alpha-Tubulin ThermoFisher #MA1-80017 (multiple lots), diluted 1:50 after conjugationCone arrestin Millipore #AB15282 (Lot: 2712407), diluted 1:100 after conjugationGFAP ThermoFisher #13-0300 (Lot: rh241999), diluted 1:50 after conjugationSV2 HybridomaBank, Antibody Registry ID: AB_2315387, in house production, diluted 1:25 after conjugationPKCα Novus #NB600-201, diluted 1:50 after conjugationCollagen IV Novus #NB120-6586, diluted 1:50 after conjugationRhodopsin EnCor Bio #MCA-A531, diluted 1:50 after conjugationCalbindin EnCor Bio #MCA-5A9, diluted 1:25 after conjugationVimentin Cell Signaling #5741S, diluted 1:50 after conjugationCalretinin EnCor Bio #MCA3G9, diluted 1:50 after conjugationVLP1 EnCor Bio #MCA-2D11, diluted 1:25 after conjugationBassoon Enzo ADI-VAM-#PS003, diluted 1:500Homer1b/c ThermoFisher #PA5-21487, diluted 1:250SupplementaryAnti-rabbit IgG (to detect Ki-67 and Homer1b/c indirectly) Jackson ImmunoResearch # 711-005-152 (Multiple lots), 1:90 afterconjugationAnti-mouse IgG (to detect Bassoon indirectly) Jackson ImmunoResearch #715-005-151) (Multiple lots), diluted 1:100 afterconjugationAnti-goat IgG (to detect Lamin B indirectly) Jackson ImmunoResearch # 705-005-147) (Lot: 125860), diluted 1:75 afterconjugationAlternative antibodies used to validate colocalization of VLP1 and Calretinin in Supplementary Fig. 8d-f:Calretinin (SantaCruz #SC-365956; EnCor Bio #CPCA-Calret; EnCor Bio #MCA-3G9 AP), VLP1 (EnCor Bio #RPCA-VLP1; EnCor Bio#CPCA-VLP1; EnCor Bio #MCA-2D11). All diluted 1:100.Fluorophore-conjugated secondary antibodies used for reference imaging:anti-rat-Alexa647 (ThermoFisher #A-21472, 1:200), anti-rabbit-Alexa488 (ThermoFisher #A-21206, 1:200), anti-rabbit-Atto488(Rockland #611-152-122S, Lot:33901, 1:500), anti-mouse-Alexa647 (ThermoFisher #A-31571, 1:400), anti-goat-Alexa647(ThermoFisher # A-21447, 1:200), anti-rabbit-Alexa647 (Jackson ImmunoResearch, 711-605-152, Lot: 125197, 1:300).Validation All antibodies used are from commercial sources as described. Only antibodies that have been validated by the vendor with in vitro and in situ experiments (for IHC and IF, with images available on the websites) and/or heavily used by the community withpublication in several references were used. The validation and references for each are publicly available on the respectivevendor websites that can reached via the catalog numbers listed above. In our experiments, IF patterns matched the distributionof cell types these antibodies were expected to label based on the literature both before and after conjugation with DNA strands. Eukaryotic cell linesPolicy information about cell linesCell line source(s)BS-C-1 cells and HeLa cellsAuthentication Cell lines were not authenticated (not relevant for the experiment or results)Mycoplasma contamination Cell lines were not tested for mycoplasma contamination (not relevant for the experiment or results)Commonly misidentified lines (See ICLAC register)No commonly misidentified cell lines were used.October 2018Animals and other organismsPolicy information about studies involving animals; ARRIVE guidelines recommended for reporting animal researchLaboratory animals Wild-type CD1 mice (male and female) age P13 or P17 were used for retina harvest.Wild animals The study did not involve wild animals.Field-collected samples The study did not involve samples collected from the field.Ethics oversight All animal procedures were in accordance with the National Institute for Laboratory Animal Research Guide for the Care and Useof Laboratory Animals and approved by the Harvard Medical School Committee on Animal Care.Note that full information on the approval of the study protocol must also be provided in the manuscript.Human research participantsPolicy information about studies involving human research participantsPopulation characteristics We have only used exempt tissue sections for technical demonstration, since we do not derive any biological conclusions, thepopulation characteristics is not relevant for this methodological study.Recruitment Not relevant for this study.Ethics oversight Human specimens were retrieved from the archives of the Pathology Department of Beth Israel Deaconess Medical Centerunder the discarded/excess tissue protocol as approved in Institutional Review Board (IRB) Protocol #2017P000585. Informedinform consent was waived on the basis of minimal risk to participants (which is indirect and not based on prospectiveparticipation by patients).Note that full information on the approval of the study protocol must also be provided in the manuscript.October 2018。

Twin-block 与肌激动器功能矫治安氏Ⅱ类下颌后缩的效果对比刘清华林俊才陈东斌蓝建灵*（福建医科大学附属龙岩市第一医院，福建龙岩364000）Factors and Improvements in Diagnosis [J].Rev Diabet Stud ，2015，12（1-2）：110-118.[5]WEI J ，ZHU A ，JI J S.A Comparison Study of Vitamin D Deficiency among Older Adults in China and the United States [J].Sci Rep ，2019，9（1）：19713.[6]RAVANI P ，MALBERTI F ，TRIPEPI G ，et al.Vitamin D levels and patientoutcome in chronic kidney disease [J].Kidney Int ，2009，75（1）：88-95.[7]GUO Y ，GUPTE M ，UMBARKAR P ，et al.Entanglement of GSK-3β，β-catenin and TGF-β1signaling network to regulate myocardial fibrosis [J].J Mol Cell Cardiol ，2017，1（10）：109-120.[8]MU M ，ZUO S ，WU R M ，et al.Ferulic acid attenuates liver fibrosis and hepatic stellate cell activation via inhibition of TGF-β/Smad signaling pathway [J].Drug Des Devel Ther ，2018，4（12）：4107-4115.[9]KIM K K ，SHEPPARD D ，CHPMAN H A.TGF-β1Signaling and Tissue Fibrosis [J].Cold Spring Harb Perspect Biol ，2018，10（4）：a022293.[10]虞建新，周理兰，许仁炜.活性维生素D 3干预糖尿病肾病大鼠模型对肾脏TGF-β1/Smads 信号轴的影响[J].中国基层医药，2017，24（18）：2760-2763.[11]ZHAO Y ，QIAO X ，TAN T K ，et al.Matrix metalloproteinase 9-dependent Notch signaling contributes to kidney fibrosis through peritubular endothelial-mesenchymal transition [J].Nephrol DialTransplant ，2017，32（5）：781-791.[12]WANG Q ，MA A ，GAO T ，et al.Poor Vitamin D Status in Active Pulmonary Tuberculosis Patients and Its Correlation with Leptin and TNF-α[J].J Nutr Sci Vitaminol （Tokyo ），2019，65（5）：390-398.[13]MAO X ，QIU J ，ZHAO L ，et al.Vitamin D and IL-10Deficiency in Preterm Neonates With Bronchopulmonary Dysplasia [J].Front Pediatr ，2018，6（11）：246.[14]ALTIERI B ，GRANT W B ，DELLA CASA S ，et al.Vitamin D and pancreas ：The role of sunshine vitamin in the pathogenesis of diabetes mellitus and pancreatic cancer [J].Crit Rev Food Sci Nutr ，2017，57（10）：3472-3488.[15]SZYMCZAK-PAJOR I ，LIWI ńSKA A.Analysis of Association between Vitamin D Deficiency and Insulin Resistance [J].Nutrients ，2019，11（4）：794.[16]MUSCOGIURI G ，CHAVEZ A O ，GASTALDELLI A ，et al.The crosstalkbetween insulin and renin-angiotensin-aldosterone signaling systems and its effect on glucose metabolism and diabetes prevention [J].Curr Vasc Pharmacol ，2008，6（4）：301-312.[17]黄映珍.2型糖尿病患者糖尿病肾脏病进展的危险因素分析[D].南方医科大学，2018.[18]MELAMED M L ，THADHANI R I.Vitamin D therapy in chronic kidney disease and end stage renal disease [J].Clin J Am Soc Nephrol （CJASN ），2012，7（10）：358-365.[19]MIZOBUCHI M ，MORRISSEY J ，FINCH J L ，et bination therapy with an angiotensin-converting enzyme inhibitor and a vitamin D analog suppresses the progression of renal insufficiency in uremic rats [J].J Am Soc Nephrol ，2007，18（6）：1796-1806.[20]LIYANAGE P ，LEKAMWASAM S ，WEERARATHNA T P ，et al.Effect of Vitamin D therapy on urinary albumin excretion ，renal functions ，andplasmareninamong patientswithdiabeticnephropathy ：A randomized ，double-blind clinical trial [J].J Postgrad Med ，2018，64（1）：10-15.[21]石天闻，周迪夷，牟新.不同剂量的活性维生素D 治疗糖尿病肾病疗效的系统评价[J].浙江医学，2018，40（1）：54-60.（收稿日期：2020-11-20）【摘要】目的对比Twin-block 与肌激动器功能矫治安氏Ⅱ类下颌后缩的效果。

ORIGINAL RESEARCHSMAD4is Involved in the Development of Endotoxin Tolerance in MicrogliaXiaorong Liu 1•Yongwei Qin 2,3•Aihua Dai 1•Yu Zhang 1•Huaqing Xue 2•Haidan Ni 1•Lijian Han 1•Liang Zhu 1•Debin Yuan 2•Tao Tao 2•Maohong Cao 1Received:10June 2015/Accepted:25August 2015/Published online:12January 2016ÓSpringer Science+Business Media New York 2016Abstract Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS,a phenomenon termed LPS tolerance.Smad4plays important roles in the induction of LPS tolerance.However,the function of Smad4in microglia remains unknown.Here we show that expression of Smad4was highly up-regulated in LPS-tolerized mouse cerebral cortex.Smad4was mostly colocalized with microglia,rarely with ing a microglia cell line,BV2,we find that LPS activates endogenous Smad4,inducing its migration into the nucleus and increasing its expression.Smad4significantly sup-pressed TLR-triggered production of proinflammatory cytokines (IL-6),increased anti-inflammatory cytokine in LPS-tolerized microglia.Moreover,IL-6concentrations in culture supernatants after second LPS challenge are higher in SMAD4small interfering RNA (siRNA)BV2cells than control siRNA BV2cells,indicating failure to induce tol-erance in absence of Smad4signaling.In our study,we conclude that both in vivo and in vitro,Smad4signaling is required for maximal induction of endotoxin tolerance.Keywords Smad4ÁMicroglia ÁEndotoxin tolerance ÁLPSIntroductionThe endotoxin LPS is a major component of the outer cell wall of gram-negative bacteria and is a potent inducer of inflammation (West and Heagy 2002).During the gram-negative bacteria infection,while detection of endotoxins by innate immune cells triggers a robust and essential inflammatory reaction,this process needs to be tightly regulated (Foster and Medzhitov 2009;Song et al.2014).But the uncontrolled inflammation leads to extensive tissue damage and manifestation of pathological states like sepsis,even septic encephalopathy.In our bodies,the protective mechanism of endotoxin is endotoxin tolerance (Biswas et al.2007;Biswas and Lopez-Collazo 2009;Dobrovol-skaia and Vogel 2002;Foster and Medzhitov 2009),a phenomenon in which cells organisms exposed to low concentrations of endotoxin (e.g.,LPS)enter into a tran-sient unresponsive state and are unable to respond to fur-ther challenges with endotoxin.This phenomenon has been observed both in vitro and in vivo (Cavaillon et al.2003;Chen et al.2012).The endotoxin tolerance of macrophage has been well studied in recently years.Microglia is a kind of macrophage of the central nervous system.Whether microglia can be induced into endotoxin tolerance is still unclear.Following infection with gram-negative bacteria,LPS,which forms a complex with LPS-binding protein,binds to the TLR4receptor complex.TLRs induce production of proinflammatory cytokines (IL-6)through MyD88and TRIF by activating MAPK and NF-j B pathways (Liu et al.2014;Wang et al.2001).IRAK-M,SHIP1,and suppressor ofXiaorong Liu and Yongwei Qin contributed equally to this work.&Tao Taonttaotao@ &Maohong Caocmhongnt@1Department of Neurology,Affiliated Hospital of Nantong University,Nantong 226001,China2Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target,Medical College of Nantong University,Nantong 226001,China3Department of Pathogen Biology,Medical College of Nantong University,Nantong 226001,ChinaCell Mol Neurobiol (2016)36:777–788DOI 10.1007/s10571-015-0260-0cytokine signaling1have been reported to be involved in the induction of endotoxin tolerance by dampening NF-j B-mediated pathway(Escoll et al.2003;Kobayashi et al.2002; Maldifassi et al.2014)(Sly et al.2004;Xiong et al.2011) (Scott et al.2009;Sun et al.2014).Recently it has been reported that Smad4is required for the development of maximal endotoxin tolerance in monocytes/macrophages (Pan et al.2010).Smad4is the common Smad and is the common mediator of signal transduction by TGF-b/BMP superfamily(ten Dijke and Hill2004).Signaling by TGF-b family members occurs through type I and type II receptors (Mu et al.2012).Activated receptor-regulated Smads form heteromeric complexes with Smad4after the activated type I receptor kinase propagates the signal inside the cell(Mu et al. 2012).Then the complexes migrate into the nucleus and interact with transcription factors,activators to induce tar-geted gene expression(ten Dijke and Hill2004).But the underlying molecular mechanism for Smad4to be involved in endotoxin tolerance is not clearly resolved.In this study,we have found that Smad4is responsible for the induction of LPS tolerance in mouse cerebral cortex and microglia.And knockdown of Smad4significantly increased the production of IL-6and decreased the pro-duction of anti-inflammatory cytokine(IL-10)in LPS-to-lerized microglia,but has no effect in wild-type microglia. Mechanically,knockdown of Smad4increased activation of NF-j B and MAPK signal pathway.Therefore,our results provide new insight to understanding the mecha-nism of endotoxin tolerance in microglia.Materials and MethodsAnimals and SurgeryAll the mice used in this study were C57BL/6J males which were provided by the Animal Center of Nantong University and between the ages of8and12weeks.The animals were housed under pathogen-free conditions in a temperature-and humidity-controlled environment and given access to food and water ad libitum.These mice were subsequentially divided into six groups including animals used for Western blot analysis,immunohistochemistry, immunofluorescence,quantitative real-time PCR(qRT-PCR),and behavior tests.To induce endotoxin tolerance, mice received1,2,or4daily intraperitoneal injections containing1.0mg/kg of LPS(E.coli;serotype055:B5; Sigma,USA)to induce endotoxin tolerance according to the previous report by Chen et al.(2012).The control groups were treated with5mg/kg LPS for similar time points but without prior injection with low-dose LPS.The subset of mice was killed24h after thefinal injection to investigate brain mRNA levels of cytokines,and the animals were anesthetized to harvest the cerebral cortex at 0,2,and4days after LPS administration for Western blotting.Then the samples were lysed in lysis buffer and stored at-80°C until use.Behavioral TestAll the experimental procedures involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals(National Research Council, 1996,USA)and were approved by the Chinese National Committee to the Use of Experimental Animals for Med-ical Purposes,Jiangsu Branch.For open-field exploration, 24h after thefinal injection,mice were placed into the center of a dimly lit(20–30lux)chamber of the open-field apparatus(44944930cm).Movements of the animals were tracked by an automatic monitoring system for 15min.Each trial lasted for3min with1trial per mouse. Time in center square,distance traveled in center square, and total distance traveled in the all areas were analyzed using Any-Maze software(Stoelting Co,Wood Dale,IL, USA).Behavioral data were analyzed by two-way ANOVA(within factor,repetition;between factor,group). Sections and ImmunohistochemistryThe animals used for immunohistochemistry were anes-thetized with Pelltobarbitalum Natricum(130mg/kg,i.p.) and perfused pericardially with0.9%saline followed by 4%paraformaldehyde.After perfusion,the brains were removed and postfixed in the samefixative for3–4days and then replaced with20%sucrose solution for2–3days, followed by immersion in30%sucrose for additional 3–4days.Tissues were then sliced into4.5-l m sections with a cryostat.All sections were stored at-20°C until use.For Smad4single immunostaining,we blocked the sections with a kind of solution consisting of10%donkey serum,1%bovine serum albumin,0.3%Triton X-100, and0.15%Tween-20for2h at room temperature and then incubated with Smad4antibody(anti-mouse,1:100,Santa Cruz)overnight at4°C,followed by incubation in biotinylated secondary antibody(Vector Laboratories, Burlingame,CA,USA).Staining was visualized with DAB (Vector Laboratories).Finally,the sections were air-dried, dehydrated,covered with coverslips,and examined using Leica microscope(Leica,DM5000B;Germany). Western Blot AnalysisIn order to prepare lysates,frozen tissues were weighed and minced on ice.Then they were homogenized in lysis buffer (50mmol/l Tris,pH7.5,5mmol/l EDTA,1%sodium deoxycholate,1%sodium dodecyl sulfate(SDS),1%Triton X-100,1%NP-40,1l g/ml leupeptin,10l g/ml aprotinin,and1mmol/l PMSF)and centrifuged at 12,000rpm at4°C for20min to collect the supernatant. After the determination of concentration with the Bradford assay(Bio-Rad),the samples were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene diflouridefilter membrane by a transfer apparatus at300mA for2h.The membrane was then blocked with5%nonfat milk and incubated with primary antibody against Smad4(anti-mouse,1:1000;Santa Cruz Biotechnology,USA),b-actin(anti-mouse;1:1000;Santa Cruz Biotechnology,USA)at4°C overnight.After incu-bating with second antibody for2h,the membrane was visualized using an enhanced chemiluminescence system (Pierce Company,USA).The sources of other Abs are as follows:rabbit anti-p38phosphorylated Abs(Cell Signal-ing Technology,USA);rabbit anti-I j B a,p50,p65,p-ERK, lamin A/C,IRAK-M,SHIP1(Santa Cruz,USA).RNA Isolation and Quantitative Real-time PCR AnalysisTotal RNA was extracted from the frozen specimens at dif-ferent survival time with Trizol Reagent(Life Technologies, Rockville,MD,USA)according to the manufacturer’s instructions.RNA was reverse transcribed with M-MLV Revert Aid TM First-Strand cDNA Synthesis Kit(Fermentas Life Sciences).qRT-PCR analysis was performed by Light Cycler(Roche Applied Science)and the SYBR RT-PCR kit (Takara).The following primers were used:mouse IL-6: forward50-CCTCTCTGCAAGAGACTTCCAT-30,reverse 50-AATTAAGCCTCCGACTTGTGAA-30,IL-1b:forward 50-GGATGAGGACATGAGCACCT-30,reverse50-AGCT CATATGGGTCCGACAG-30,TNF-a:forward50-ACAGA AAGCATGATCCGCGA-30,reverse50-GTTTGCTACGA CGTGGGCT-30on a MyIQ real-time PCR detection system (Bio-Rad).PCR amplification was performed with an initial denaturing step at95°C for5min and subsequently carried out for40cycles,with each cycle consisting of denaturation at94°C for10s,primer annealing at65°C for15s,and extension at72°C for15s.The reaction wasfinally incu-bated for one cycle at72°C for10min.Double-Immunofluorescence StainingFor double staining,brain sections were incubated with primary antibodies specific for smad4(mouse;1:100;Santa Cruz Biotechnology,USA),NeuN(rabbit;1:100;ABclonal Biotechnology,USA),GFAP(rabbit;1:100;Sigma,USA), and CD11b(rabbit;1:100;Millipore,USA)at4°C over-night,followed by a mixture of FITC-and TRITC-conju-gated secondary antibodies(Jackson ImmunoResearch)for 2h at4°C.The stained sections were viewed under a Leicafluorescence microscope(Leica,DM5000B;Leica CTR5000;Germany).The cells grown on coverslips were fixed with4%formaldehyde for30min,subsequently treated with0.1%Triton X-100in phosphate-buffered saline(PBS)for5min,and blocked with PBS containing 3%normal goat serum for1h.Then,the cells were incubated overnight at4°C with anti-Smad4antibodies, anti-Iba-1antibodies,and DAPI,washed three times with PBS,and incubated with FITC-and TRITC-conjugated secondary antibodies(Jackson ImmunoResearch)for2h at 4°C.Cells were examined under a Leicafluorescence microscope(Leica,DM5000B;Leica CTR5000;Ger-many).Cells double labeled for Smad4and the other phenotypic marks used in the experiment were quantified. To identify the proportion of each phenotype-specific marker-positive cell expressing Smad4,a minimum of200 phenotype-specific marker-positive cells was counted in each section.For all the experiments,two or three inter-mittent sections per animal sections per animal were sampled.Cell Cultures and StimulationBV2is a murine microglial cell line immortalized with v-raf/v-myc genes carrying retrovirus J2.Cells were cul-tured in DMEM with10%FBS.Mixed glial cultures were prepared from cerebral cortices of1-day-old C57BL/6 mice(Charles River,France)according to the method of Giulian et al.(1986).After mechanical and chemical dis-sociation,cortical cells were seeded in DMEM-F12with 10%FBS at a density of250,000cells/ml(62,500cells/ cm2)and cultured at37°C in humidified5%CO2/95% air.Medium was replaced every4–5days and confluency was achieved after10–12days in vitro(DIV).Microglial cultures were prepared by the mild trypsinization method. Mixed glial cultures were incubated with a trypsin solution (0.25%trypsin,1mM EDTA in HBSS;named henceforth trypsin0.25%)diluted1:4in DMEM-F12resulted in the detachment of an upper layer of cells in one piece,whereas a number of cells remained attached to the bottom of the well.The detachment was typically completed after 25–35min.Twenty-four hours after trypsinization,pri-mary microglia were treated with LPS(1l g/ml).These cells were used in experiments to evaluate the role of Smad4in endotoxin tolerance.For tolerance, microglia were pretreated with0.1l g/ml LPS from E.coli for18h,rested in fresh media for2h,and then restimu-lated with LPS as indicated(Peng et al.2012).Cytokine DetectionCells were harvested at a specified period,and supernatants were stored for ELISA analysis.IL-10and IL-6weremeasured with ELISA kits (ExCell Bio,Shanghai,Chi-na).The OD of each well was read by using a microplate reader at 450nm with 540-nm correction (BioTek,USA).Each sample was executed in triplicates.Each experiment was repeated at least three times.Cell TransfectionOligo sequences used were as follows:shSMAD4-RNA,50-GCAGCAGAAUGGAUUUACUTT-30.For transient transfection,the SMAD4siRNA vector and the control vector were carried out using lipofectamine2000(Invitro-gen)and plus reagent in OptiMEM (Invitrogen)as sug-gested by the manufacturer.Transfected cells were used for the subsequent experiments 24h after transfection.Immunoblot and Nuclear and Cytoplasmic ExtractionCells were lysed with cell lysis buffer supplemented with protease inhibitor mixture.Protein concentrations of the extracts were measured with the Bradford assay (Bio-Rad,Hercules,CA).Cells (19107)in 75cm 2plates,at conflu-ency,were washed with ice-cold PBS and suspended in 200l l of hypotonic buffer [10mM HEPES (pH 7.9),10mM KCl,0.1mM EDTA,0.1mM EGTA,1mM DTT,1mM PMSF,and a protease inhibitor cocktail].The cells were allowed to swell on ice for 15min;then,2.5l l of 10%NP-40was added to the cell suspension.The cell suspension was agitated on a vortex for 10s and then centrifuged at 2000rpm for 5min.The resulting supernatant is represented as the cytosolic extract.The pellets containing the nucleus were resuspended in 50l l of ice-cold nuclear extraction buffer [20mM HEPES (pH 7.9),400mM NaCl,1mM EDTA,1mM EGTA,1mM DTT,0.5mM PMSF,and aprotease inhibitor cocktail]and incubated on ice for 1h with intermittent vortexing.The pellet thus treated was cen-trifuged at 15,000rpm for 10min,and the resulting super-natant is represented as the nuclear fraction.The protein sample was stored at -80°C before use.Statistical AnalysisAll data were performed with Stata 8.0statistical software.All values are expressed as mean ±SEM and were ana-lyzed by one-way variance (ANOVA)followed by Tukey’s post hoc multiple comparisons tests.P \0.05or P \0.01was considered statistically significant.Each experiment consisted of at least three replicates precondition.ResultsEndotoxin Tolerance Can be Induced in Microglia To know whether endotoxin tolerance can be induced in microglia,BV2cells were treated with low-dose LPS (0.1l g/ml)for 18h,rested in fresh media for 2h,and then restimulated with 1l g/ml LPS for 0,3,6,and 12h.This activation protocol is referred as ‘‘tolerized.’’Control cells were treated with 1l g/ml LPS for similar time points but without prior stimulation with low-dose LPS.This activa-tion protocol is referred as ‘‘untolerized’’(Hu et al.2014;Pan et al.2010).ELISA results showed that IL-6decrease significantly in low-dose LPS-primed cells compared with unprimed cells (Fig.1a),and IL-10increase over time in low-dose LPS-primed cells compared with unprimed cells (Fig.1b).These data indicated that in the microglia,endotoxin tolerance can be induced by low-dose LPSpretreatment.Fig.1Microglia can be induced into endotoxin tolerance.BV2cells were treated with 1or 0.1–1l g/ml of LPS for 0,1,3,6,and 12h.After specific period,cell culture supernatants were assessed for IL-6(a )and IL-10(b )concentration detection by ELISA.Each treatment was executed in triplicates.Results are shown as mean ±SD of three independent experiments.*P \0.05Expression of Smad4in Microglia After LPS ChallengeTo further explore the role of Smad4in the development of endotoxin tolerance,we detected the expression of Smad4 in microgliafirstly.The Smad4protein level was signifi-cantly induced,with even higher expression in tolerized microglia compared with untolerized microglia(Fig.2a, b).In further,we have detected the Smad4levels in nuclear and cytosol.Smad4was increased in the nuclear and reached the peak at6h in tolerized microglia,but it was decreased gradually at12h in untolerized groups(Fig.2c, d).But in the cytosol,Smad4was decreased in tolerized microglia and increased in untolerized groups(Fig.2c,d). These results indicated that in the endotoxin tolerance of microglia,Smad4is activated following LPS stimulation. Knockdown of Smad4Failure to Induced Endotoxin Tolerance in MicrogliaTaking into account the vigorously inducible expression of Smad4,Smad4was knockdown by siRNA.The knock-down of Smad4was efficiency after6h in the secondary LPS challenge,in which time point the induction of Smad4expression was most vigorous(Fig.3a).After knockdown smad4expression,the IL-6and IL-10were not signifi-cantly affected in the untolerized BV2(Fig.3b,c).How-ever,knockdown of Smad4increased TLR4-induced production of IL-6and decreased IL-10in LPS-tolerized microglia after the second LPS stimulation(Fig.3c),which is consistent with others(Pan et al.2010).To further confirm the function of inducible Smad4in LPS-tolerized BV2cells model,we observed the effects of Smad4 silencing on MAPK and NF-j B pathways activation in LPS-tolerized BV2cells.After transfected with control siRNA and Smad4siRNA,BV2cells were induced toler-ized or only treated with one LPS injection for0,10,30, and60min.p-p38,p-ERK,and p-p65levels were sus-tained or slightly decreased in Smad4knockdown BV2 cells following second exposure to LPS,whereas they decreased considerably with second LPS treatment in control siRNA BV2cells(Fig.3d,e).LPS-induced degradation of total I j B a protein was enhanced in Smad4 knockdown BV2compared with control siRNA BV2cells following second exposure to LPS.These results demon-strate that p-p38,p-ERK,and p-p65continue to be acti-vated in tolerized Smad4knockdown BV2cells.We investigated whether Smad4also regulates SHIP1and Fig.2Smad4participates in endotoxin tolerance development.a BV2cells were treated with1or0.1–1l g/ml of LPS for0,1,3,6,and12h.After specified period,cells were harvested.Western blotanalysis was used to detect the Smad4expression.b Data werenormalized to the relative expression of the b-actin.c Cytosolic andnuclear proteins were isolated from the above-treated cells andsubjected to Western blot analysis.All blots were reprobed with anti-b-actin to show equal loading.d Quantification graphs(relativeoptical density)of the intensity of a ratio of nuclear to cytosolic ofSmad4at each time point.Data are presented as mean±SD of threeindependent experiments(*P\0.05)Fig.3Knockdown of Smad4by Smad4-siRNA results in increased NF-j B activation in BV2cells.a Immunoblot assay of the Smad4protein expression level in BV2cells transfected with Smad4-specific siRNA (Smad4-Si)or scramble control (Ctrl-Si).ELISA of IL-6(b)and IL-10(c)cytokine production in supernatants from LPS-tolerized (0.1l g/ml)BV2cells with Smad4silencing or non-silencing,and restimulated with a secondary round of LPS(1l g/ml)as indicated.Data are representative of three independent experiments with similar results or are shown as mean±SD of three independent experiments.*P\0.05.d Immunoblot analysis with the indicated antibodies of cell lysis from BV2transfected with Smad4-specific siRNA(Smad4-Si)or scramble control(Ctrl-Si), tolerized with LPS(0.1l g/ml), and restimulated with a second round of LPS(1l g/ml)as indicated.Data are representative of three independent experiments with similar results.e Quantification graphs(relative optical density) of the intensity of the staining of I j B a,p-p65,p-p38,p-ERK at each time point.Data are presented as mean±SD(n=3,*,#,%,&P\0.05,significantly different from the control siRNA-treated group).f Isolated proteins from Si-Smad4and control siRNA BV2 cells were subjected to Western blot analysis with the indicated Abs.Blots were reprobed with anti-b-actin to show equal loading.h Data are presented as mean±SD(n=3,*,#,&P\0.05,significantly different from the control siRNA-treated group)IRAK-M in tolerized cells.Endogenous Smad4was knocked down with siRNA.We found that both SHIP1and IRAK-M protein expressions were reduced in Smad4knockdown BV2cells compared with control siRNA BV2cells (Fig.3f,g).Smad4Participates in Endotoxin Tolerance in Primary CellsTo confirm the induction of Smad4in endotoxin tolerance of primary cells,Smad4was knockdown by siRNA (Fig.4A).Mouse primary microglia were treated with 1l g/ml or 0.1–1l g/ml LPS for 0,10,30,and 60min.IL-6secretion was significantly higher in the Smad4knock-down primary microglia cells than in the control primary microglia cells in both untolerized and tolerized primary microglia cells (Fig.4b).By contrast,IL-10secretion was significantly decreased in the Smad4knockdown primary microglia compared with the control siRNA primary microglia (Fig.4c).These results further demonstrate that Smad4is a negative regulator of IL-6.In further,we also detected the activation of MAPK and NF-j B signal path-way in primary microglia.The degradation of I j B a wasalso more vigorous in Smad4-silenced primary microglia cells,which led to more activation of the transcription factor NF-j B.Knockdown of Smad4increased the acti-vation of p-p38,p-ERK,and p-p65(Fig.4d).Smad4Expression was Increased in LPS-tolerized Mouse Cerebral CortexTo further confirm the role of Smad4in microglia endo-toxin tolerance,we used mice model to define the role in vivo.Mice were peripheral administered 1.0mg/kg LPS for 4consecutive days to induce endotoxin tolerance and subsequently injected with 5mg/kg LPS according to the previous report by Marsh et al.(2009)and Weberpals et al.(2009).Control groups were treated with 5mg/kg LPS for similar time points but without prior injection with low-dose LPS.Open-field testing has assessed general motor behavior of the mice including distance and mean speed in a particular period of time (Kolber et al.2010).There was a general tendency of the LPS-tolerized mice to set longer distances than the untolerized mice.Also,the speed of LPS-tolerized mice was faster than the untolerized mice (Fig.5a).Assessment of TNF-a ,IL-6,IL-1b ,andIL-10Fig.4The expression changes of Smad4,I j B a ,and p65after LPS injection in primary cells.Smad4gene of the mouseprimary microglia was knocked down with Smad4siRNA,and control siRNA was taken as control (a ).Mouse primary microglia were treated with 0.1l g/ml LPS for 18h,then rested in fresh media for 2h,and restimulated with a second round of LPS (1l g/ml)for 0,1,3,6,and 12h.After the indicated periods,the culture supernatants were assessed by ELISA for IL-6(b )and IL-10(c )concentration.Each treatment was executed intriplicates.Each experiment was repeated at least three times.Results shown are themean ±SD.*P \0.05.d The above cells were harvested for Western blot analysis with the indicated Abs.The blots were reprobed with anti-b -actin to show equal loading.Results are representative of the three experimentsexpression by qRT-PCR in mouse brain showed that pre-treatment with continuous 1mg/kg LPS injections for 2or 4days significantly down-regulated the production of IL-6compared with the non-pretreated groups (Fig.5b).These data indicated that in the mouse brain,the endotoxin tol-erance can be induced.Based on that,we explored Smad4expression mouse cerebral cortex.The protein level of Smad4reached the peak at 2day after LPSstimulation.Fig.5Establishment of endotoxin tolerance in animal models and Smad4expression in cerebral cortex.Mice were left treated with LPS for 4consecutive days to induce endotoxin tolerance and subse-quently injected with 5mg/kg LPS (tolerized).The control groups were treated with 5mg/kg LPS for similar time points but without prior injection with low-dose LPS (untolerized).a Open-field testing assessed general motor behavior of the mice including the normal group,the untolerized group,and the tolerized group.Both the untolerized and tolerized animals were the fourth day after final injection.Error bars represent SEM of the three independent experiments.*P \0.05.b The minus means the mice receive no LPS injection.The plus means the mice receive daily LPS injections as the time indicated.qRT-PCR analysis of the mRNA expression level of TNF-a ,IL-6,IL-1b ,and IL-10in cerebral cortex as indicated.Data are representative of three independent experiments with similar results or are shown as mean ±SD of three independent experiments.*P \0.05,#P \0.05,&P \0.05,%P \0.05.c Western blot of Smad4protein expression in both untolerized and tolerized groupsAfter4days of continuous injection of LPS,the mice were reinjected with the second round of LPS.The protein level of Smad4was significantly induced,with even higher expression(Fig.5c).Smad4May be Involved in Microglia Endotoxin Tolerance In VivoTo identify staining changes and distribution of Smad4after LPS injection,we performed immunohistochemistry stain-ing on transverse cryosections of brain cortex.The number and intensity of Smad4-positive cells increased markedly in the cerebral cortex of LPS-tolerized group,compared with the cerebral cortex of LPS-untolerized group(Fig.6a).No staining signal was observed in the negative control (Fig.6a).Quantitative analysis documented that there was a dramatic elevation of Smad4-positive cells in LPS-tolerized group(Fig.6b;P\0.05).These results implied that the temporal pattern of Smad4after LPS injection was consistent with the results of Western blot.In order to further study Smad4cellular localization in the brain after endotoxin tol-erance,double-labeling immunofluorescent was performed in transverse cryosections of brain tissues with cell specific markers:NeuN(a marker of neuron),GFAP(a marker of astrocyte),and CD11b(a marker of microglia),to distinguish different cell types.Smad4was expressed in neurons (Fig.6c),microglia(Fig.6c);meanwhile,Smad4was localized mainly in microglia(Fig.6c).Quantitative analy-sis reflected that Smad4was mainly located in microglia cells(Fig.6d;P\0.05).These data indicated Smad4may be involved in endotoxin tolerance in mouse model. DiscussionPrior exposure of innate immune cells like macrophages to minute amounts of endotoxin causes them to become refractory to subsequent endotoxin challenge;this phe-nomenon we called‘‘endotoxin tolerance.’’Clinically,this state is associated with monocytes/macrophages in sepsis patients and may contribute to‘‘immunosuppression’’and mortality.After bacteraemia,septic patients suffer from a refractory state which is similar to endotoxin tolerance (Cavaillon and Adib-Conquy2006;Monneret et al.2008). Circulating monocytes isolated from septic patients show characteristics of endotoxin tolerance which indicate the importance of an endotoxin-tolerant mechanism.In vitro and in vivo models of endotoxin tolerance have been used in several studies(Escoll et al.2003;Lopez-Collazo et al. 2006;Manjuck et al.2000;Pachot et al.2006).Up to now, there are very few effective approaches verified to address endotoxin tolerance.In this study,we investigated whether Smad4,one of the LPS-inducible genes,was involved in sepsis encephalopathy,and microglia endotoxin tolerance. We have demonstrated that Smad4is important in the maintenance of the reduced inflammatory cytokine pro-duction in LPS-tolerized microglia by inhibiting NF-j B signaling pathway.Previous study has demonstrated the role of microglia in brain LPS tolerance.A single intraperitoneal injection of LPS transiently protects against ischemic infarcts but does not activate microglia or protect against cryogenic-induced cortical injury;on the other hand,four intraperitoneal injections of LPS confer protection against traumatic brain injury but not ischemic stroke.Thus,it appears that four LPS injections may induce neuroprotection against trau-matic brain injury(Chen et al.2012).On the basis of the findings,we connected the protection with endotoxin tol-erance in microglia.So we focused on whether Smad4was involved in endotoxin tolerance in microglia cells.Smad4is probably induced by TLR4signaling as well as autocrine production of TGF-b.It has been shown that stimulation of THP1cells with LPS or TGF-b may result in induction of Smad4.Smad4is a negative regulator of IL-6. TGF-b may negatively regulate LPS signaling through Smad4during endotoxin tolerance development in both THP-1cells and primary mouse cells(Pan et al.2010).But the role of Smad4in microglia in CNS is still unclear.We have found Smad4was highly up-regulated in LPS-tolerant mouse cerebral cortex which was located in microglia.The significant up-regulation of Smad4was also observed in LPS-stimulated mouse microglia,which indicated that Smad4might be involved in sepsis encephalopathy,and microglia endotoxin tolerance.Also,the expression levels of Smad4and anti-inflammatory proteins IL-10were up-regulated upon stimulation with1l g/ml or0.1–1l g/ml LPS within a period of12h.The abrogation of Smad4 expression resulted in higher level of IL-6release and low level of anti-inflammatory proteins IL-10following1l g/ ml or0.1–1l g/ml LPS stimulation compared with control cells,which indicates a partial failure of induction of endotoxin tolerance and highlights the critical role of Smad4signaling in this phenomenon.The second exposure to LPS leads to the reduced phosphorylation of p38and ERK in control siRNA cells,but not in Smad4siRNA BV2 cells(Fig.3d).Total I j B a degradation is much faster in Smad4siRNA BV2cells than in control siRNA cells. Knockdown of Smad4increased nuclear translocation of transcriptors p65(Fig.3d).Thus,Smad4negatively regu-lates LPS signaling through inhibiting NF-j B signaling pathway.Taken together,p38and p-ERK are activated in BV2cells(untolerized)upon thefirst exposure to LPS,and p38and p-ERK are inactivated in LPS-restimulated cells (tolerized).P38and ERK activations are retained in both untolerized and tolerized Smad4siRNA cells,along with higher IL-6production.。

87M.A. Hayat (ed.), Stem Cells and Cancer Stem Cells, Volume 6,DOI 10.1007/978-94-007-2993-3_9, © Springer Science+Business Media B.V . 20129A bstractRetinal and macular degeneration disorders are characterized by a progressive loss of photoreceptors, which causes visual impairment and blindness. In some cases, the visual loss is caused by dysfunction, degen-eration and loss of underlying retinal pigment epithelial (RPE) cells and the subsequent death of photoreceptors. The grim reality is that there is no successful treatment for most of these blindness disorders. Cell therapy aimed at replenishing the degenerating cells is considered a potential ther-apeutic approach that may delay, halt or perhaps even reverse degenera-tion, as well as improve retinal function and prevent blindness in the aforementioned conditions. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) may serve as an unlimited donor source of photoreceptors and RPE cells for transplantation into degenerat-ing retinas and for retinal disease modeling.I ntroductionThe vertebrate eyes form as bilateral evaginations of the forebrain, called optic vesicles (Martínez-Morales et al. 2004 ; Fig. 9.1a ). During develop-ment, the optic vesicles begin to invaginate to form a cup-shaped structure, the optic cup. The inner, thicker neural layer of the optic cup differ-entiates into the neural retina, and the outer, thin-ner pigmented layer forms the retinal pigmentepithelium (RPE). At the early developmental stages, the neuroepithelial cells that compose the optic vesicle are morphologically and molecu-larly identical and are all able to give rise to neu-ral retina and RPE. Exogenous signals coming from the adjacent tissues, including factors from the fi broblast growth factor (FGF) and transform-ing growth factor beta (TGF b ) families, dictate the fate of these cells. The mature vertebrate ret-ina is comprised of six types of neurons and one type of glia (the Müller glia). These seven cell types constitute three nuclear layers: retinal gan-glion cells in the ganglion cell layer (GCL); the horizontal, bipolar and amacrine interneurons, and Müller glial cells in the inner nuclear layer (INL); and rod and cone photoreceptors in the outer nuclear layer (ONL; Harada et al. 2007;M . I delson • B . R eubinoff (*)T he Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy & The Department of Obstetrics and Gynecology , H adassah University Medical Center ,E in Kerem 12000 ,J erusalem 91120 ,I srael e -mail: b enjaminr@ekmd.huji.ac.il D ifferentiation of HumanPluripotent Stem Cells into Retinal Cells Masha Idelson and Benjamin Reubinoff88M. Idelson and B. ReubinoffFig. 9.1b ). The photoreceptor cells capture lightphotons and transform their energy into electrical signals by a mechanism called phototransduction. The visual pigment which is utilized in this process is located on membranal discs in the outer seg-ments of photoreceptors. The outer segments are continuously renewed: the old discs are shed and new disks form. When the photoreceptors absorb light, they send the signal through the retinal interneurons to the ganglion cells which transmit the electrical impulse to the brain by their axons forming the optic nerve. Rods are responsible for night vision, whereas cones are responsible for color vision and detecting fi ne details. The macula is a small part of the retina which is rich in cones and responsible for detailed central vision.R PE cells that compose the outer layer of the optic cup are pigmented cuboidal cells which lie between the neural retina and the choriocapil-laris, which include the blood vessels supplying the retina. The multiple villi on their apical side are in direct contact with the outer segments ofextraocular mesenchymeabneural retinalensoptic nerveoptic cupsurface ectodermRPEFGFoptic vesiclechoroidBM RPE cone ONLINL GCLlightHC BC MC ACONrod F ig. 9.1 D evelopment and structural arrangement of the retina. ( a ) Schematic representation of retinal development including the transition from optic vesicle to optic cup and retinal patterning. ( b ) Schematic diagram of retinal cells arrangement and connections. A bbreviations :A C amacrinecell, B C bipolar cell, B M Bruch’s membrane, G CL gan-glion cell layer, H C horizontal cell, I NL inner nuclear layer, M C Müller cell, O N optic nerve, O NL outer nuclear layer89 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsthe photoreceptor cells; on their basal side, the RPE is in contact with the underlying basal mem-brane, termed Bruch’s membrane that separates the RPE from the choroid. These cells play cru-cial roles in the maintenance and function of the retina and its photoreceptors. As a layer of pig-mented cells, the RPE absorbs the stray light that was not absorbed by the photoreceptors. The RPE cells form a blood–retinal barrier due to decreased permeability of their junctions. The RPE cells transport ions, water, and metabolic end products from the retina to the bloodstream. They are involved in supplying the neural retina with nutrients from the bloodstream, such as glu-cose, retinol, and fatty acids. Another important function of the RPE is the phagocytosis of shed photoreceptor outer segments. After the outer segments are digested, essential substances such as retinal are recycled. Retinal is also recycled and returned to photoreceptors by the process known as the visual cycle. The precise functioning of the RPE is essential for visual performance. Failure of one of these functions can lead to degeneration of the retinal photoreceptors, vision impairment and blindness.T here are many inherited and age-related eye disorders that cause degeneration of the retina as a consequence of loss of photoreceptor cells. Retinal and macular degeneration disorders can be divided into two main groups. The fi rst group primarily affects the photoreceptors and involves the majority of cases of retinitis pigmentosa. In the second group, the primary damage is to the adjacent RPE cells, and as a consequence of this damage, the photoreceptors degenerate. This group includes age-related macular degeneration, Stargardt’s macular dystrophy, a subtype of Leber’s congenital amaurosis in which RPE65 is mutated, Best’s disease and some cases of retini-tis pigmentosa, as well.W ith regard to retinitis pigmentosa (RP), it is a group of inherited retinal degeneration diseases that are caused, as mentioned above, by a primary progressive loss of rod and cone photoreceptors, followed by a subsequent degeneration of RPE (Hartong et al. 2006). The disease affects approxi-mately 1.5 million patients worldwide and is the most common cause of blindness in people under 70 years of age in the western world. The disease can be characterized by retinal pigment deposits visible on the fundus examination. In most cases, the disease primarily affects rods. At later stages of the disease, the degeneration of cones takes place. As a consequence of disease progression, the patients’ night vision is reduced. Patients initially lose peripheral vision while retaining central vision (a visual status termed “tunnel vision”). In advanced cases, central vision is also lost, commonly at about 60 years of age. The disease affects about 1 in 4,000. The inheritance can be autosomal-recessive, autosomal-dominant or X-linked (in ~50–60%, 30–40%, and 5–15% of cases, respectively). Mutations in more than 140 genes have been iden-tifi ed as causing RP (Hartong et al. 2006).Among these genes are those involved in phototransduc-tion, like rhodopsin, the a- and b- subunits of phos-phodiesterase, the a- and b- subunits of Rod cGMP gated channel and arrestin. The additional muta-tions were found in genes encoding structural pro-teins, like peripherin, rod outer segment protein and fascin. They were also found in transcription factors involved in photoreceptors’ development such as Crx and Nrl, and in other genes, whose products are involved in signaling, cell-cell interac-tion and trafficking of intracellular proteins. Currently, there is no effective cure for RP. Treatment with vitamin A palmitate, omega-3 fatty acids and other nutrients may somewhat slow the rate of the disease progression in many cases. Reduction in exposure to light was also shown to decrease the rate of retinal degeneration.A mong the group of retinal degenerations that are caused by primary loss of RPE cells or their function, age-related macular degeneration (AMD) is the most frequent condition and the leading cause of visual disability in the western world (Cook et al. 2008).Among people over 75 years of age, 25–30% are affected by AMD, with progressive central visual loss that leads to blindness in 6–8%. The retinal degeneration pri-marily involves the macula. The dry form of AMD is initiated by hyperplasia of the RPE and formation of drusen deposits, consisting of meta-bolic end products underneath the RPE or within the Bruch’s membrane. It may gradually progress into the advanced stage of geographic atrophy90M. Idelson and B. Reubinoff with degeneration of RPE and photoreceptorsover large areas of the macula causing central visual loss. Ten percent of dry AMD patients will progress to neovascular (wet) AMD, with blood vessels sprouting through the Bruch’s membrane with subsequent intraocular leakage and/or bleed-ing, accelerating the loss of central vision. While the complicating neovascularization can be treated with anti-VEGF agents, currently there is no effective treatment to halt RPE and photore-ceptor degeneration and the grim reality is that many patients eventually lose their sight (Cook et al. 2008).S targardt’s macular dystrophy (SMD) is the most common form of inherited macular dystro-phy affecting children (Walia and Fishman 2009). The disease is symptomatically similar to AMD. The prevalence of SMD is about 1 in 10,000 chil-dren. The disease involves progressive central visual loss and atrophy of the RPE beneath the macula following accumulation of lipofuscin in RPE cells, which is suggested to consist of non-degradable material, derived from ingested pho-toreceptor outer segments. The inheritance is predominantly autosomal recessive, although an autosomal dominant form has also been described. The mutation in the ABCA4 gene was found to be a most common cause of SMD. The product of the ABCA4 gene is involved in energy transport to and from photoreceptors. The mutated protein cannot perform its transport function and, as a result, photoreceptor cells degenerate and vision is impaired. Currently, there is no effective treat-ment for SMD.C ell therapy to replenish the degenerating cells appears as a promising therapeutic modality that may potentially halt disease progression in the various retinal and macular degeneration dis-orders caused by loss and dysfunction of RPE cells and photoreceptors (da Cruz et al. 2007).I n this chapter we will discuss the potential of human pluripotent cells which includes human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs), to gen-erate various types of retinal cells that could be used for transplantation therapy of retinal degen-eration disorders and disease modeling for drug discovery. C ell Therapy of Retinal and Macular DegenerationsT he eye is an attractive organ for cell therapy as it is easily accessible for transplantation and for simple monitoring of graft survival and potential complications by direct fundoscopic visualiza-tion. Anatomically, it is a relatively confi ned organ limiting the potential of unwanted extra-ocular ectopic cell distribution, and a low number of cells are required to replenish the damaged cells. The eye is also one of the immune privi-leged sites of the body.T he concept of replacing dysfunctional or degenerated retina by transplantation has been developing ever since the fi rst retina-to-retina transplant in 1986 (Turner and Blair 1986).In most studies, primary retinal immature (fetal) tissue has been used as donor material. It was demonstrated that such transplants can survive, differentiate, and even establish connections with the host retina to a limited degree (Ghosh et al. 1999). The subretinal transplantation of healthy RPE has some advantages over neural retinal transplantation, as it concerns only one cell type that is not involved in neural networking. Transplantation of RPE has been studied exten-sively in animal models (Lund et al. 2001).The most commonly used animal model of retinal degeneration is the Royal College of Surgeons (RCS) rat model, in which primary dysfunction of the RPE occurs as a result of a mutation in the receptor tyrosine kinase gene M ertk(D’Cruz et al. 2000). This leads to impaired phagocytosis of shed photoreceptor outer segments, with sec-ondary degeneration and progressive loss of pho-toreceptors within the fi rst months of life. It was reported that rat and human RPE cells rescued photoreceptor cells from degeneration when transplanted into the subretinal space of RCS rats (Li and Turner 1988; Coffey et al. 2002).The ability of transplanted RPE cells to restore retinal structure and function has been demonstrated in clinical trials. In humans, autologous transplanta-tions of peripheral RPE as well as macular trans-locations onto more peripheral RPE provide a proof that positioning the macula above relatively91 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellshealthier RPE cells can improve visual functionin AMD patients (Binder et al. 2004; da Cruz et al. 2007). Nevertheless, the surgical procedures for autologous grafting are challenging and are often accompanied by signifi cant complications. In addition, autologous RPE transplants may carry the same genetic background, environmen-tal toxic and aging-related effects that may have led to macular RPE failure and the development of AMD in the patient. It is also problematic to use autologous cells when all the RPE cells are damaged. Cell sources that can be used for such therapy include allogeneic fetal and adult RPE (Weisz et al. 1999; Binder et al. 2004; da Cruz et al. 2007). However, the use of fetal or adult retinal tissues for transplantation is severely lim-ited by ethical considerations and practical prob-lems in obtaining sufficient tissue supply. The search for a cell source to replace autologous RPE such as immortalized cell lines, umbilical cord-derived cells as well as bone marrow-derived stem cells continues.T he derivation of hESCs more than a decade ago has raised immense interest in the potential clinical use of the cells for regeneration (Thomson et al. 1998; Reubinoff et al. 2000).Along the years, signifi cant progress has been made towards the use of hESCs in clinical trials.T he other promising source of cells for transplantation therapy is iPSCs that are simi-lar to hESCs in their stemness characteristics and pluripotency. These cells could be gener-ated from different human somatic cells by transduction of four defi ned transcription fac-tors: Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al. 2007).G eneration of RPE and neural retina from hESCs and iPSC has numerous advantages, as it can be done from pathogen-free cell lines under good manufacturing practice (GMP) conditions with minimal variation among batches. Such cells can be characterized extensively prior to preclinical studies or for clinical applications, and an unlimited numbers of donor cells can be generated from them. In the following para-graphs, strategies for induction of differentiation of hESCs and iPSCs towards RPE and neural retina fate are reviewed. D ifferentiation into Retinal Pigment EpitheliumI t was reported for the fi rst time in mice and pri-mates that the differentiation of ES cells into RPE could be induced by co-culture with PA6 stromal cells (Kawasaki et al. 2002; Haruta et al. 2004). The resulting cells had polygonal epithelial mor-phology and extensive pigmentation. The cells expressed the markers that are characteristic of RPE. They developed typical ultrastructures and exhibited some functions of RPE. The differenti-ation of hESC into RPE was first reported by Klimanskaya et al. (2004).According to their protocol, hESCs underwent spontaneous differ-entiation by overgrowth on mouse embryonic fibroblasts (MEF), in feeder-free conditions or, alternatively, as embryoid bodies (EBs) in com-bination with withdrawal of bFGF from the medium. The yield of the formation of RPE cells after 4–8 weeks of spontaneous differentiation was relatively low; for example,<1% of EBs con-tained pigmented cells at this stage. However, after 6–9 months in culture, all the EBs contained pigmented cells. The areas of pigmented cells could be further isolated mechanically and prop-agated by passaging as RPE lines. Klimanskaya and colleges characterized the hESC-derived RPE cells by transcriptomics and demonstrated their higher similarity to primary RPE tissue than to human RPE lines D407 and ARPE-19. The low yield of spontaneously differentiating RPE cells was improved by induction of differentia-tion with Wnt and Nodal antagonists, Dkk1 and LeftyA, respectively, the factors that are sug-gested to promote retinal differentiation. This treatment gave rise to pigmented cells within 38% of the hESC colonies after 8 weeks (Osakada et al. 2008). Immunostaining with the ZO-1 anti-body showed that by day 120, hESC-derived pig-mented cells formed tight junctions (about 35% of total cells). We showed that differentiation toward the neural and further toward the RPE fate could be augmented by vitamin B3 (nicotin-amide; Idelson et al. 2009).We further showed that Activin A, in the presence of nicotinamide, effi ciently induces and augments differentiation92M. Idelson and B. Reubinoffinto RPE cells. This is in line with the presumed role of Activin A in RPE development i n vivo .In the embryo, extraocular mesenchyme-secreted members of the TGF b superfamily are thought to direct the differentiation of the optic vesicle into RPE (Fuhrmann et al. 2000).Under our culture conditions, when the cells were grown in suspen-sion as free-fl oating clusters, within 4 weeks of differentiation, 51% of the clusters contained pigmented areas and about 10% of the cells within the clusters were pigmented. When we modifi ed the differentiation conditions to includea stage of monolayer culture growth, the yield of the RPE-like pigmented cells was signifi cantly improved and 33% of the cells were pigmented after 6 weeks of differentiation. The derivation of RPE from hESCs and iPSCs without any external factor supplementation was also demonstrated by other groups (Vugler et al. 2008 ; Meyer et al. 2009 ; Buchholz et al. 2009).T he hESC-derived RPE cells were extensively characterized, including demonstration, both at the mRNA and the protein levels, of the expres-sion of RPE-specifi c markers, such as RPE65, CRALBP, Bestrophin, Tyrosinase, PEDF, PMEL17, LRAT, isoforms of MiTF abundant in RPE, and others. The cells expressed markers of tight junctions that join the adjacent RPE cells: ZO-1, occludin and claudin-1 (Vugler et al. 2008 ) . Electron microscopic analysis revealed that the hESC-derived RPE cells showed features characteristic of RPE. The cells were highly polarized with the nuclei located more basally, and the cytoplasm with the mitochondria and melanin granules of different maturity more api-cally. A formation of basal membrane was observed on the basal surface of the RPE cell. Similar to putative RPE, the hESC-derived RPE basal membrane was shown to be composed of extracellular matrix proteins, collagen IV , lami-nin and fi bronectin (Vugler et al.2008).The appearance of apical microvilli was demonstrated at the apical surface of the RPE. The presence of tight and gap junctions on the apical borders of the RPE cells was also confi rmed by electron microscopy. O ne of the most important functions of RPE cells i n vivo is phagocytosis of shed photoreceptor outer segments, as part of the continuous renewal process of rods and cones. The hESC-derived RPE cells demonstrated the ability to phagocyto-size latex beads or purifi ed photoreceptor outer segments, confi rming that these cells are func-tionali n vitro . It may be concluded from all these studies that human pluripotent stem cells have a potential to give rise to pigmented cells exhibiting the morphology, marker expression and functionof authentic RPE.D ifferentiation into Retinal Progenitors and Photoreceptors O ur group showed, for the fi rst time, the potential of highly enriched cultures of hESC-derived neu-ral precursors (NPs) to differentiate towards the neural retina fate (Banin et al. 2006).We demon-strated that the NPs expressed transcripts of key regulatory genes of anterior brain and retinal development. After spontaneous differentiation i n vitro , the NPs gave rise to progeny expressing markers of retinal progenitors and photoreceptor development, though this was uncommon and cells expressing markers of mature photorecep-tors were not observed. We showed that after transplantation into rat eyes, differentiation into cells expressing specifi c markers of mature photoreceptors occurred only after subretinal transplantation (between the host RPE and pho-toreceptor layer) suggesting that this specifi c microenvironment provided signals, yet unde-fi ned, that were required to support differentia-tion into the photoreceptoral lineage.P rogress towards controlling and inducing the differentiation of hESCs into retinal progenitors and neurons i n vitro was reported in the study of Lamba et al. ( 2006).They treated hESC-derived EBs for 3 days with a combination of factors,including Noggin, an inhibitor of BMP signaling, Dkk1, a secreted antagonist of the Wnt signaling pathway and insulin-like growth factor 1 (IGF-1), which is known to promote retinal progenitor dif-ferentiation. The cultivation of EBs with these factors was followed by differentiation on Matrigel or laminin for an additional 3 weeks in the presence of the combination of the three93 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsfactors together with bFGF. Under these culture conditions, the majority of the cells developed the characteristics of retinal progenitors and expressed the specifi c markers Pax6 and Chx10 (82% and 86% of the cells, respectively). The authors showed that after further differentiation, the cells expressed markers of photoreceptor development Crx and Nrl (12% and 5.75%, respectively). About 12% of the cells expressed also HuC/D, the marker of amacrine and ganglion cells. The expression of markers of the other sub-types of retinal neurons was demonstrated, as well. However, only very few cells (<0.01%) expressed markers of mature photoreceptors, blue opsin and rhodopsin. The abundance of cells expressing markers of photoreceptors could be accelerated by co-culture with retinal explants, especially when the explants originated from mice bearing a mutation that causes retinal degeneration.T o better characterize the phenotype of retinal cells obtained with this differentiation protocol, a microarray-based analysis comparing human retina to the hESC-derived retinal cells was per-formed (Lamba and Reh 2011).It was demon-strated that gene expression in hESC-derived retinal cells was highly correlated to that in the human fetal retina. In addition, 1% of the genes that were highly expressed in the hESC-derived cultures could be attributed to RPE and ciliary epithelium differentiation.A n alternative protocol for the derivation of retinal progenitors and photoreceptors was pro-posed by Osakada et al. (2008).Similar to the protocol for the derivation of RPE cells, they used serum-free fl oating cultures in combination with the Dkk1 and LeftyA. After 20 days of cul-ture in suspension, the cells were replated on poly-D-lysine/laminin/fi bronectin-coated slides. Osakada and co-authors demonstrated that on day 35 in culture, about 16% of colonies were positive for retinal progenitor markers Rx and Pax6. Differentiation towards photoreceptor fate was augmented in the presence of N2 by treat-ment with retinoic acid and taurine, which are known inducers of rod fate differentiation. Under these conditions, after an extended culture period of 170 days, about 20% of total cells were positive for Crx, an early photoreceptor marker. On day 200, about 8.5% of the cells expressed the mature rod photoreceptor marker, rhodopsin, as well as cone photoreceptor markers, red/green and blue opsins (8.9% and 9.4%, respectively).A n alternative approach was proposed by the same group based on the use of small molecules. In this method, the chemical inhibitors CKI-7 and SB-431542 that inhibit Wnt and Activin A signaling, respectively, and Y-27632, the Rho-associated kinase inhibitor, which prevents disso-ciation-induced cell death, were used. These molecules were shown to mimic the effects of Dkk1 and LeftyA (Osakada et al. 2009).This strategy, which doesn’t involve the use of recom-binant proteins which are produced in animal or E scherichia coli cells, is more favorable for the gen-eration of cells for future transplantation therapy.I n another study that was published by Meyer et al .(2009), after initial differentiation in sus-pension for 6 days, the aggregates were allowed to attach to laminin–coated culture dishes. After further differentiation as adherent cultures, neu-roepithelial rosettes were formed, which were mechanically isolated and subsequently culti-vated as neurospheres. The authors didn’t use any soluble factors; moreover, they showed that under these conditions, the cells expressed endogenous Dkk1 and Noggin. They also demonstrated that in concordance with the role of bFGF in retinal specifi cation, the inhibition of endogenous FGF-signaling abolished retinal differentiation. Under their differentiation protocol, by day 16, more than 95% of the cells expressed the retinal pro-genitor markers, Pax6 and Rx. The authors dem-onstrated that by day 80 of differentiation, about 19% of all neurospheres contained Crx+ cells and within these Crx+ neurospheres, 63% of all cells express Crx and 46.4% of the cells expressed mature markers, such as recoverin and cone opsin.I n all of the above studies, differentiated cells expressing the retinal markers were obtained; however, the cells were not organized in a three-dimensional retinal structure. In a paper recently published by Eiraku et al. (2011),the authors cul-tured free-fl oating aggregates of mouse ES cells in serum-free medium in the presence of base-ment membrane matrix, Matrigel, that could also94M. Idelson and B. Reubinoffbe substituted with a combination of laminin, entactine and Nodal. Using a mouse reporter ES cell line, in which green fl uorescent protein (GFP) is knocked in at the Rx locus, the authors showed that Rx-GFP+ epithelial vesicles were evaginated from the aggregates after 7 days of differentiation under these conditions. On days 8–10, the Rx-GFP+ vesicles changed their shape and formed optic cup-like structures. The inner layer of these structures expressed markers of the neural retina whereas the outer layer expressed markers of RPE. The authors demonstrated that differen-tiation into RPE required the presence of the adjacent neuroectodermal epithelium as a source of diffusible inducing factors. In contrast, the differentiation into neural retina did not require tissue interactions, possibly because of the intrinsic inhibition of the Wnt-signaling pathway. Eiraku and colleagues showed that the retinal architecture, which was formed within the optic vesicle-like structures, was comparable to the native developing neural retina.R ecently, optic vesicle-like structures were also derived from hESCs and iPSCs using the protocol described above, which is based on iso-lating the neural rosette-containing colonies and culturing them in suspension (Meyer et al. 2011). The cells within the structures expressed the markers of retinal progenitors, and after differen-tiation gave rise to different retinal cell types. It was shown that the ability of optic vesicle-like structures to adopt RPE fate could be modulated by Activin A supplementation. The production of these three-dimensional retinal structures opens new avenues for studying retinal development in normal and pathological conditions.T ransplantation of Pluripotent Stem Cell-Derived Retinal CellsA key step towards future clinical transplanta-tions of hESC-derived RPE and neural retina is to show proof of their therapeutic potential i n vivo. Various animal models of retinal degeneration have been used to evaluate the therapeutic effect of transplanted retinal cells. Human ESC-derived RPE cells were transplanted subretinally to the degenerated eyes of RCS rats. Transplantation of the hESC-derived RPE cells between the RPE and the photoreceptor layer rescued retinal struc-ture and function (Lund et al. 2006; Vugler et al. 2008; Idelson et al. 2009; Lu et al. 2009).The subretinally engrafted hESC-derived RPE cells salvaged photoreceptors in proximity to the grafts as was shown by the measurement of the thick-ness of the ONL, the layer of photoreceptor nuclei, which is an important monitor of photore-ceptor cell survival. The ONL thickness was significantly increased in transplanted eyes in comparison to the degenerated non-treated eyes.I n order to evaluate the functional effect of transplanted cells i n vivo, the electroretinography (ERG) that directly measures the electrical activ-ity of the outer (a-wave) and inner (b-wave) retina in response to light stimulation was used. It was demonstrated that after transplantation of hESC-derived RPE, ERG recordings revealed a signifi -cant preservation of retinal function in the treated eyes as compared to control untreated eyes (Lund et al. 2006; Idelson et al. 2009).The visual func-tion of the animals was also estimated by an optomotor test, which monitors the animal’s refl exive head movements in response to a rotat-ing drum with fi xed stripes. Animals transplanted with hESC-derived RPE showed signifi cantly better visual performance in comparison to con-trol animals (Lund et al. 2006; Lu et al. 2009). The presence of rhodopsin, a major component of photoreceptor outer segments, within the sub-retinaly transplanted pigmented cells suggested that they could perform phagocytosis i n vivo (Vugler et al. 2008; Idelson et al. 2009).B ridging the gap between basic research and initial clinical trials requires immense resources to ensure safety and efficacy. Human ESC-derived RPE cell lines were generated using a current Good Manufacturing Practices (cGMP)-compliant cellular manufacturing process (Lu et al. 2009). Long-term studies analyzing safety and efficacy of transplantation of these GMP-compliant hESC-derived RPE cells revealed that the subretinally transplanted cells survived for a period of up to 220 days and provided prolonged functional improvement for up to 70 days after transplantation. The potential of the hESC-derived。

qScript ® Ultra Flex KitDescriptionThe qScript Ultra Flex Kit is a complete, next-generation system that delivers rapid and efficient first-strand cDNA synthesis while providing flexibility in the choice of priming method. The kit features a stabilized 5x-concentrated reaction mix that provides all components for first-strand synthesis except RNA template and primers. A key component is a novel, state-of-the-art, RNase H deficient reverse transcriptase (RT) that was engineered for improved thermostability, velocity, processivity, and resistance to many common reaction inhibitors.The superior performance of this novel RT is further supported by proprietary replication accessory proteins and a recombinant mammalian RNase inhibitor protein. These features allow for reactions to be carried out at higher temperatures than standard reverse transcriptases, improving sensitivity and minimizing potential interference from blocking secondary structures. In addition, the improved synthesis speed and enhanced processivity allow for reactions to be complete in 10 minutes, with high yields of full-length cDNA products as long as 20 kb.The kit is supplied with both an anchored oligo(dT) solution and a modified random primer mixture that enhances cDNA yield with low input quantities or compromised RNA samples. Primer solutions include a proprietary enhancer compound that improves cDNA priming efficiency and protects RNA integrity during the optional denaturation step to destabilize RNA secondary structures. This enhancer is provided as a separate solution for use with your gene-specific primers. The kit is compatible with total RNA, polyA+ RNA, or viral RNA. The resulting cDNA product is directly compatible with real-time RT-qPCR methods or end-point RT-PCR. The length of cDNA product is dependent on the priming strategy and the quality of the RNA template. Greater length cDNA products can be obtained from oligo(dT) priming, in which primers specifically anneal to the 3’ poly(A) tails of mRNA, or by using gene-specific priming, in which primers anneal to a defined sequence. While strategies that utilize random primers produce shorter first strand cDNA, they are suitable for obtaining larger yields of product from the 5’-ends of RNA molecules and from classes of RNA biotypes that do not contain a 3’ poly(A) tail. Complete, unbiased first strand cDNA synthesis can be obtained with a mixed primer strategy, in which oligo(dT) and random primers are combined.Components95215-025 95215-100 5x qScript Ultra Reaction Mix1 x 100 µL1 x 400 µLOptimized master mix containing buffer, magnesium, dNTPs and qScript Ultra RTOligo(dT) primers1 x 50 µL1 x 200 µL10x concentrated in Enhancer solutionRandom primers1 x 50 µL1 x 200 µL10x concentrated in Enhancer solutionGSP Enhancer (10x) 1 x 50 µL 1 x 200 µL Nuclease-free Water1 x 1.5 mL2 x 1.5 mLCat No. 95215-025 Size: 25 x 20 µL reactions (1x 0.1 mL) Store at -25ºC to -15°C95215-100 100 x 20 µL reactions (1x 0.4 mL)Storage and StabilityStore components in a constant temperature freezer at -25°C to -15°C.After thawing, mix thoroughly before use.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form. Standard Reaction Protocol1.Thaw components, mix and centrifuge before use. Hold on ice before use.2.Add the following to a thin-walled PCR tube or reaction plate on ice:Component Volume for 20 μL rxn.Final Concentration Nuclease-free water variableTemplate RNA variable 2.5 µg to 1 pg total RNA10x Oligo(dT) or 10X Random Primers or 1-10 µM Gene-specific primer(s) 2 µL1x Oligo(dT) or 1x Randomprimers or 0.1-1 µM Gene-specific primers(Optional) 10x GSP Enhancer, if using Gene-specificprimer(s)2 µL5x qScript Ultra Reaction Mix 4 µLFinal volume 20 µLNOTE: For multiple reactions, a master mix can be prepared with all components except template RNA and dispensed into 96-well plates or PCR tubes.3.Mix by gentle vortexing, then briefly centrifuge to collect contents.4.Incubate for:o 5-10 minutes at 25°C (only required if using random primers)o 10 minutes at 55°Co 5 min at 85°Co Hold at 4°C5.After the completion of cDNA synthesis, reactions can be used directly for endpoint RT-PCR or RT-qPCR analysis. It isrecommended that PCR reactions contain no more than 1/5 volume of the first-strand cDNA reaction. If desired, reactions can be diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Reaction can be stored at -20°C for future use. Two-Phase Reaction ProtocolFor improved yield of some long cDNA products, or if template is known to contain regions of secondary structure1.Thaw components, mix and centrifuge before use. Hold on ice before use.2.Add the following to a thin-walled PCR tube or reaction plate on ice:Component Volume for 20-μL rxn.Final ConcentrationNuclease-free water variableTemplate RNA variable 2.5 µg to 1 pg total RNA10x Oligo(dT) or 10X Random Primers or 1-10 µM Gene-specific primer(s) 2 µL1X Oligo(dT) or 1X Randomprimers or 0.1-1 µM Gene-specific primers(Optional) 10x GSP Enhancer, if using Gene-specificprimer(s)2 µL Final volume 16 µL3.Mix by gentle vortexing, then briefly centrifuge to collect contents.4.Incubate for 5 min at 65°C then immediately transfer to 4°C.5.Add to each reaction mixture:Component Volume for 20-μL rxn.Final Concentration5x qScript Ultra Reaction Mix 4 µL 1XFinal Volume (μL) 20 µL6.Mix by gentle vortexing, then briefly centrifuge to collect contents.7.Incubate for:o 5-10 minutes at 25°C (only required if using random primers)o 10 minutes at 55°Co 5 min at 85°Co Hold at 4°C8.After the completion of cDNA synthesis, reactions can be used directly for endpoint RT-PCR or RT-qPCR analysis. It isrecommended that PCR reactions contain no more than 1/5 volume of the first-strand cDNA reaction. If desired, reactions can be diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Reaction can be stored at -20°C for future use.Quality ControlFunctional PCR Assay for Flex Kit Reaction Mix (5X): Detection of ß-actin mRNA from 100 ng to 100 fg of total RNA. Coefficient of determination (R2 ) ≥ 0.990 with a slope analysis between -3.20 and -3.70. Single bands visible at 9 kb and 15 kb from 30 cycles of PCR using 100 ng Oligo-dT cDNA.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use withcomponents contained in the kit only. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio, LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rightsof third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate anyacts prohibited above. Quantabio, LLC. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915Telephone number: 1-888-959-5165.Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, GermanyIntended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.TrademarksqScript and ToughMix are registered trademark of Quantabio, LLC.。

在胚胎组织和胚胎发育过程中Hedgehog（HH）通路具有非常显著的作用，如增殖、分化、和组织构建等，在成年人的组织和器官中，HH信号传导涉及组织修复和再生。

作为对靶基因进行调控的核心因子，Gli能够对TGF-β通路进行调控。

对于多种细胞过程，转化生长因子-β（TGF-β）家族因子都能够进行调控，其中包括细胞外基质合成、细胞迁移、分化和增殖等，对于组织内环境稳定的维持和胚胎发育都能够实现积极的调控作用。

通过对下游Smad蛋白进行介导，依赖Gli2实现对Gli1的快速诱导，进而实现对HH通路的调控。

尽管它们的典型信号转导级联已被很好地表征，但越来越多的证据表明这些途径能够发挥重叠的活性，成为纤维化和癌症的治疗靶向。

1. HH信号通路的调控作用下游靶基因、转录因子、跨膜蛋白和膜蛋白复合物以及分泌性糖蛋白配体是组成HH通路的主要基本内容。

Desert HH（Dhh）、Indian HH（Ihh）和Sonic HH（Shh）这三种分泌型Hh配体是哺乳动物最基本的三种配体，并且这三种配体所在区域各不相同。

在毛发、皮肤、肢体、消化道和神经系统当中主要是SHH进行参与；在胰腺、消化道的发育过程中主要是Ihh进行参与；外神经周围、胰腺和性腺当中才会有Dhh分布。

其中阳性表达最高、分布最广的蛋白就是Shh。

HH信号通路中存在两个跨膜蛋白受体：Smoothened （Smo）和Ptched（Ptch）是，Ptch1与Ptch2分别是Ptch的两个同源基因。

Hh配体与Ptch 的结合活性受到生长阻滞特异基因1（growth arrestspecific gene1，Gas1）、Hh相互作用蛋白（Hip）、细胞黏附分子相关/被癌基因下调基因（cell adhesion molecule- related/ down- regulated by oncogenes，Cdon/ Cdo）及其家族成员Boc等的调节。

Hip抑制Hh与Patch结合，负性调控HH信号通路。

Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制杨俊侠;曹述任;张敏【摘要】目的:观察Smad泛素化调节因子2(Smurf2)在转化生长因子β1(TGF-β1)诱导人肺成纤维细胞活化中的作用,并探讨其可能的分子机制.方法:体外培养人胚肺成纤维细胞MRC-5,10 μg·L-1 TGF-β1作用1、2和6h(分别为TGF-β1 1 h组、TGF-β1 2 h组和TGF-β1 6 h组),并设对照组(不加TGF-β1),RT-PCR和Western blotting法分别检测各组细胞中Smurf1和Smurf2 mRNA和蛋白的表达水平.MRC-5细胞随机分为对照组(未加入TGF-β1或siRNA)、TGF-β1组(10 μg·L-1 TGF-β1)、Control siRNA转染组(10 μg·L-1 TGF-β1+Control siRNA)和Smurf2 siRNA转染组(10 μg·L-1 TGF-β1+ Smurf2 siRNA).Western blotting法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原a1 (COL1A1)的表达水平;RT-PCR和Western blotting法分别检测各组细胞中Smad7、核转录共抑制因子SnoN、TGF-β Ⅰ型受体(TβR Ⅰ)、Smad2和Smad3 mRNA和蛋白的表达水平.结果:与对照组比较,各TGF-β1组细胞中Smurf2 mRNA和蛋白表达水平均明显升高(P＜0.05),且随着TGF-β1作用时间的延长,其表达水平呈逐渐增加的趋势;与对照组比较,各TGF-β1组细胞中Smurf1表达水平无明显变化(P＞0.05).与TGF-β1组比较,Smurf2 siRNA组细胞中Smurf2蛋白表达水平下降(P＜0.05).与对照组比较,TGF-β1组细胞中α-SMA和COL1A1蛋白表达水平均明显升高(P＜0.05);与TGF-β1组比较,Smurf2 siRNA组细胞中α-SMA和COL1A1蛋白表达水平均下降(P＜0.05).与对照组比较,TGF-β1组和Smurf2 siRNA组细胞中Smad7和SnoN mRNA表达水平均升高(P＜0.05),而Smurf2 siRNA组和TGF-β1组细胞中Smad7和SnoN mRNA表达水平无明显变化(P＞0.05).与对照组比较,TGF-β1组细胞中Smad7和SnoN蛋白表达水平均明显下降(P＜0.05);与TGF-β1组比较,Smurf2 siRNA组细胞中Smad7和SnoN蛋白表达水平明显升高(P＜0.05).与对照组比较,TGF-β1组和Smurf2 siRNA组细胞中TβRⅠ mRNA和蛋白表达水平均明显升高(P＜0.05),而Smurf2 siRNA组和TGF-β1组细胞中TβRⅠ mRNA和蛋白表达水平比较差异无统计学意义(P＞0.05).各组细胞中Smad2和Smad3 mRNA和蛋白表达水平比较差异无统计学意义(P＞0.05).结论:Smurf2可能通过泛素化降解Smad7和SnoN,参与调控TGF-β1/Smads信号通路,从而发挥其促进TGF-β1活化肺成纤维细胞的作用.【期刊名称】《吉林大学学报（医学版）》【年(卷),期】2015(041)005【总页数】7页(P891-897)【关键词】Smurf2;肺成纤维细胞;转化生长因子β1;信号转导【作者】杨俊侠;曹述任;张敏【作者单位】暨南大学第四附属医院广东省广州市红十字会医院呼吸内科,广东广州510220;暨南大学第四附属医院广东省广州市红十字会医院呼吸内科,广东广州510220;暨南大学第四附属医院广东省广州市红十字会医院呼吸内科,广东广州510220【正文语种】中文【中图分类】R563Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制杨俊侠，曹述任，张敏（暨南大学第四附属医院广东省广州市红十字会医院呼吸内科，广东广州510220）［摘要］目的：观察Smad泛素化调节因子2（Smurf2）在转化生长因子β1（TGF－β1）诱导人肺成纤维细胞活化中的作用，并探讨其可能的分子机制。