人角膜缘干细胞的体外原代培养和生物学鉴定

目的　建立人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 养液对人角膜缘干细胞进行体外培养,进行形态学观察和免疫细胞化学鉴定。结果　植块3～4d后细胞开始贴壁 生长,10～15d形成良好的单层,细胞呈圆形、卵圆形,类角膜上皮细胞;免疫细胞化学染色显示,培养第11d,少量 细胞表达AE5,大量细胞表达p63。结论　应用20%胎牛血清1640培养液组织培养的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。

Primary Culture and Biological Characterization of Human Corneal Limbal Stem Cells in Vitro

Objective To establish a method for culturing human 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. The cultured cells were identified immunochemically by two monoclonal antibodies including AE5 and p63. Results Stem cells cultured in FBS grew well with the adhesion of the wall within 3~4 days and formed a confluent layer in 10~15 days, the primary isolated cells in shape were similar to corneal epithelial cells with round or oval shape. Immunochemical staining showed that most human cultured cells were p63 positive and only a few cells AE5 positive on the 11th day. Conclusions The limbal stem cells could be successfully cultured in 20% FBS culture medium and the cultured cells have the same characteristics of limbal stem cells.