人胎儿角膜缘干细胞的体外培养及鉴定

目的 探讨人胎儿角膜缘干细胞的体外培养及鉴定方法。方法 应用消化培养法对人胎儿角膜缘上皮组织进行体外培养并观察记录培养细胞的生长特性；对胎儿角膜缘组织及培养细胞采用一系列单克隆抗体进行免疫酶细胞化学染色检测；并用扫描电镜观察原代细胞的表面状况。结果 胎儿角膜缘上皮细胞原代培养时生长旺盛，传代培养时保持高增殖速率。免疫酶细胞化学染色见培养前胎儿角膜缘组织基底层有多量AE5阴性、AE1和PCNA阳性细胞，并见较多HLA-DR阳性细胞；原代细胞绝大部分AE5和HLA-DR染色阴性，AEI和PCNA阳性；传代培养至第3代时AE5阴性细胞仍占大部分；扫描电镜观察原代细胞以球形或短圆柱形为主，表面多突起和微绒毛。结论 采用消化培养法可成功培养出具有高增殖力和低抗原性的人胎儿角膜缘干细胞．为临床眼表重建开辟了新的思路。

Ex Vivo Culture and Identification of Human Fetal Limbal Stem Cells

Objective To investigate techniques of culture and identification of human fetal lim- bal stem ceils ex vivo. Methods Human fetal limbal epithelial tissue was cultured in vitro by enzymatic digestion culture method and the morphological characteristics of cultured cells were recorded. The fetal limbus and cultured cells were detected by enzyme immunocytochemistry staining with a series of monoclonal antibodies. The surface characteristics of primary culture cells were observed by scanning electron microscope. Results Fetal limbal epithelial ceils grew swiftly in primary culture and kept high proliferating rate in subculture. In the results of enzyme immunocytochemistry staining, many cells in basal layer of fetal limbus were AE5 negative, AE1, PCNA and HLA-DR positive. A absolute majority of primary culture ceils were AE5 and HLA-DR negative, AE1 and PCNA positive. Moreover, a majority of ceils in the third passage were AE5 positive. The morphological characteristics of primary culture cells were mainly spherical shape or short column shape and had plenty of processes and microvilli on surface by scanning electron microscope. Conclusion Human fetal limbal stem cells with high proliferating ability and lower antigenicity can be cultured successfully by enzymatic digestion culture method, so they may show broad applying prospect in clinical ocular surface reconstruction.