兔角膜缘干细胞两种培养方法的比较  

Comparison of two different culture techniques of rabbit limbal stem cells

作　　者：张晓敏[1] 孙慧敏[1] 赵少贞[1] 李筱荣[1] 孙靖[1] 袁佳琴[1] 

机构地区：[1]天津医科大学眼科中心,300070

出　　处：《眼科研究》2005年第6期575-578,共4页Chinese Ophthalmic Research

摘　　要：目的对采用消化法和组织块法培养兔角膜缘干细胞进行对比,以期确定合理的角膜缘干细胞体外培养方法。方法采用3T3成纤维细胞滋养层培养体系,分别用消化法和组织块法培养兔角膜缘上皮细胞,用免疫组织化学技术检测培养的细胞中ΔNp63的表达。采用流式细胞技术分析分离的角膜缘上皮细胞悬液中间质细胞的含量,采用扫描电镜观察去除上皮的角膜缘基质表面。结果采用消化法培养,培养的细胞中ΔNp63表达较多,细胞最终可以分化为复层上皮结构。在分离的细胞悬液中,间质细胞含量小于5%,扫描电镜显示角膜缘上皮细胞能被完全消化。结论在角膜缘干细胞的培养中,在干细胞的含量方面,消化法培养要明显优于组织块法培养。Objective To compare cell-suspension technique with explant technique in the culture of rabbit limbal stem cells. Methods With feeder layer culture system, cell-suspension method and explant method were used in the culture of limbal epithelial cells. Expression of ΔNp63 in cultured cells was detected using immunohistochemistry and vimentin, and cytokeratin expressions were assessed by flow eytometric analysis. Content of mesenchymal cells in the cell-suspension was deteeted by flow cytometrie analysis, and limbal stroma surface was examined by scanning electron microscope to assess cell-suspension method. Results In single layer cells cultured by cell-suspension technique,there were much more cells expressing ΔNp63. Epithelial cells cultured with cell-suspension method could eventually differentiated into stratified epithelium. Mesenchymal cells content obtained in the cell-suspension was less than 5%. Scanning electron microscope examination revealed that intact limbal epithelium sheet could be isolated from limbal tissue. All the limbal epithelial cells,including basal cells, were removed and only basement membrane with an irregular surface is visible. Comby structure on the irregular surface was consistent with irregular cytoplasmie extensions of basal cells. Conclusion In view of stem cells content in the culture system, cell-suspension technique is superior to explants technique.

关 键 词：兔 角膜缘干细胞 体外培养 ΔNP63 

分 类 号：R77[医药卫生—眼科]