体外兔胚胎成纤维细胞为饲养层克隆兔角膜缘干细胞

目的研究兔胚胎成纤维细胞体外克隆兔角膜缘干细胞。方法兔胚胎成纤维细胞经丝裂霉素C(MMC)处理成饲细胞,用消化法培养兔角膜缘基底层细胞并接种于含有经MMC处理饲细胞的12孔培养板,进行原代培养。观察其光镜特征、电镜结构,用间接免疫细胞化学染色等对克隆的细胞进行综合鉴别。结果克隆的细胞呈典型的上皮细胞形态,电镜下可见微绒毛、桥粒、张力丝等典型上皮细胞结构,单克隆抗体AE1、PCNA染色后细胞大多数阳性,单克隆抗体AE5染色后细胞染色偶见阳性。结论体外兔胚胎成纤维细胞为饲养层成功地克隆出兔角膜缘干细胞。

In vitro clone of rabbit corneal limbal stem cells supporting by rabbit embryo fibroblast feeder cells

ObjectiveTo investigate the properties of rabbit corneal limbal stem cells cloned by rabbit embryo fibroblast feeder cells in vitro.MethodsRabbit embryo fibroblast was treated by Mitomycin C to become feeder cells.Rabbit corneal limbal basal cells was cultured primarily by enzymatic digestion and inoculated in 12-well culture plate with feeder cells.Cultured cells were analyzed under the light microscopy,scanning and transmission electron microscopy.Indirect immunecytochemistry staining was used to identify the cultured cells comprehensively.ResultsCultured cells were cloned at the 2nd day and formed a monolayer on 10-12 days and showed a typical epithelium-like with a large quantity of microvilli,desmosomes and tonofilaments under the electron microscopy.Rabbit corneal limbal stem cells cloned with feeder cells demonstrated a potential passage activity as compared with those without feeder cells.Most cloned cells showed a positive staining for monoclonal antibodies AE1 and PCNA and minority of the cells was positive for AE5.ConclusionRabbit corneal limbal stem cells can be cloned successfully from rabbit embryo fibroblast feeder cells and maintain corneal epithelial cell characteristics and high proliferative potency.