・组织工程・
小肠黏膜下层复合上皮细胞及成肌细胞构建
组织工程食管的初步研究
岑石强1 李万里1 黄富国1 杨志明2 解慧琪2 罗静聪2
【摘 要】 目的 初步探索利用小肠黏膜下层(s m all in testinal subm uco sa,S IS)作为支架材料,复合食管鳞状上皮细胞与成肌细胞构建组织工程食管的可行性。 方法 4周龄无胸腺小鼠20只,体重20.0±2.5g,雌雄不限。体外分离、培养人胚胎食管鳞状上皮细胞、成肌细胞,并行52B rdU标记;制备S IS材料并切割成1c m×1c m大小,在S IS材料同一表面接种两种细胞,待细胞在材料表面贴附后将其植入裸鼠体内深筋膜下,于术后第3天及1、2、3周取材行组织学和抗角蛋白、平滑肌肌动蛋白(s moo th m u scle actin,S M A)免疫组织化学观察,以了解食管鳞状上皮细胞、成肌细胞在S IS 材料上的增殖、分化情况和复合物在裸鼠体内的血管化情况。 结果 人胚胎食管鳞状上皮细胞、成肌细胞能够渗透、生长于S IS材料中,并形成数层结构。植入体内第3天可见细胞增殖为多层覆盖于材料表面,并分泌大量细胞间基质。第2周时已经形成7～8层细胞,并伴有血管长入。3周时细胞增殖为十几层,大量血管长入。通过52B rdU标记抗体染色观察,示S IS支架材料上生长的细胞多为所植入细胞。抗角蛋白,S M A免疫组织化学染色示移植细胞能够在体内分化。 结论 成肌细胞与鳞状上皮细胞在S IS材料上的复合培养物,在体内能继续增殖分化形成多层细胞结构,并能快速血管化,可用于组织工程化食管的构建。
【关键词】 组织工程食管 小肠黏膜下基质 食管鳞状上皮细胞 成肌细胞
中图分类号:R318.08 Q813 文献标识码:A
PREL I M INAR Y RESEARCH ON CONSTRUCTI ON OF ARTIF I C I AL ES OPHAGUS W ITH CUL TURED SQUA MOUS EP ITHEL I AL CELL S AND MYOB LAST CELL S SEED ED ON S M ALL INTESTINAL SUB M UCOSA CEN S h iqiang, L I W an li,H UA N G F ug uo,et a l.D ep a rt m en t of O rthop ed ics,W est Ch ina H osp ita l,S ichuan U n iversity,Cheng d u S ichuan,610041,P.R.Ch ina
Corresp ond ing au thor:YA N G Z h i m ing,E2m a il:orthop@p
【Abstract】 Objective To study the con structi on feasib ility of a b i odegradab le artificial esophagu s by the squamou s ep ithelial cells and the m yob last cells seeded on the s m all in testinal subm uco sa(S IS)and to investigate the grow th pattern and angi ogenesis of the co2cu ltu red hum an em b ryon ic squamou s ep ithelial cells and the skeletal m yob lasts in v ivo.M ethods T he squamou s ep ithelial cells and the m yob last cells w ere ob tained from the202w eek abo rted fetu s.Bo th of their cells w ere m arked by52B rdU in v itro.T he iso lated cells w ere then seeded on the S IS and co2 cu ltu red in v itro fo r24hou rs,and then the compound of the cells and the S IS w as tran sp lan ted in to the subcu taneou s tissue of the athym is m u s m ice.T he ob servati on on the mo rpho logy and the cytokeratin A E3andΑ2actin specified
i m m unoh istochem istry of the squamou s ep ithelial cells and the m yob last cells w as perfo rm ed at each of the fo llow ing
ti m e po in ts:3days,1w eek,2w eek s,and3w eek s after tran sp lan tati on.Results T he mo rpho logical ob servati on indicated that the cu ltu red cells cou ld penetrate in to the s m all in testinal subm uco sa and fo rm several2layered cell structu res,and that the compound of the cells and the S IS cou ld have angi ogenesis w ith in223w eek s.T he52B rdU specified i m m unoh istochem ical ob servati on suggested that the cells grow ing in the s m all in testinal subm uco sa scaffo ld m igh t be the cells tran sp lan ted.T he cytokeratin A E3specified andΑ2actin specified i m m unoh istochem ical studies demon strated that the tran sp lan ted cells cou ld differen tiate in v ivo.Conclusion It is po ssib le to fab ricate the fram ew o rk of a b i odegradab le artificial esophagu s w ith the ep ithelial cells and the m yob last cells seeded on the s m all in testinal subm uco sa.
【Key words】 T issue engineered esophagu s Sm all in testinal subm uco sa E sophgu s squamou s ep ithelial cells M yob last cells
Founda tion ite m:N ati onal N atu ral Science Foundati on of Ch ina(30100182)
基金项目:国家自然科学基金资助项目(30100182)
作者单位:1四川大学华西医院骨科(成都,610041),2生物治疗国家重点实验室・干细胞与组织工程研究室
通讯作者:杨志明,教授,博士导师,研究方向:组织工程,E2m ail:o rthop@
