兔角膜缘干细胞的体外原代培养和生物学鉴定

Primary Culture of Rabbit Corneal Limbal Stem Cells and Its Biological Differentiation in Vitro

目的建立兔角膜缘干细胞体外培养方法,观察其生物学特性.方法用含20%胎牛血清1640培养液对兔角膜缘干细胞进行体外培养,倒置显微镜观察培养细胞体外生长的形态,电镜观察培养细胞的超微结构并进行免疫细胞化学和间接免疫荧光鉴定.结果植块48～72 h细胞开始贴壁生长,7～10 d形成良好的单层,细胞呈圆形、卵圆形或多边形,类似角膜上皮细胞;电镜下,可见细胞表面有许多微绒毛,细胞间有桥粒连接,胞质丰富,胞质细胞器较多,含有线粒体等结构;免疫细胞化学和间接免疫荧光染色显示,培养第8天少量细胞角蛋白K3(AE5)免疫反应呈阳性,细胞质被染色,培养第14天和第21天,较多细胞AE5免疫反应阳性.结论含20%胎牛血清1640培养液能作为角膜缘干细胞培养的基础液.应用组织培养方法获得的原代培养细胞具有与正常角膜缘干细胞相一致的生物学特性.

Purpose To establish a method for culturing rabbit limbal stem cell in vitro and observe its biological characteristics. Methods Corneal limbal stem cells were cultured in medium containing 20% fetal bovine serum (FBS).And the morphology of the monolayer-cultured cells was observed by inverted phase contract microscope.Ultrastructure of cell was observed by electron microscopy.The cultured cells were identified by immunochemically and immunofluorescence staining by monoclonal antibody AE5,which recognized 64 000 keratin. Results Stem cells cultured in FBS adhered and grew within 48～72 hours and formed a confluent layer in 7～10 days,the shape of the primary isolated cells were similar to corneal epithelial cells.There were a lot of microvilli on their surface and desmosones connected between the cultured cells.And there were many mitochondria in the cells.The rabbit cultured cells were a few positive for cytokeratin AE5 monoclone antibody on the 8 th day,and there were more positive cells on the 14th and 21st day. Conclusions Twenty percent FBS culture medium can be used as the basical medium for the stem cell culture.The limbal stem cells could be successfully cultured in vitro and own the same characteristics as normal limbal stem cells have.