人表皮干细胞的体外分离培养和鉴定

Isolation,culture and identification of hmnan epithelial stem cells

目的:探讨人表皮干细胞的体外快速分离培养及鉴定方法。方法:中性蛋白酶和胰蛋白酶两步法从手术切除的人包皮组织中分离表皮层和真皮层,并获得表皮单细胞悬液,采用Ⅳ型胶原铺板选择性粘附、分离和角质形成细胞无血清培养基(K-SFM)培养表皮干细胞。倒置显微镜下观察培养细胞的生长状况,检测细胞克隆形成率,免疫组化染色观察表皮干细胞标志物β1整合素和角蛋白19(K19)的表达;以角质形成细胞作为对照。结果:组织学观察显示,培养24h后细胞呈克隆状生长;所分离、培养细胞的克隆形成率高于对照角质形成细胞组;免疫组化染色显示,培养细胞β1整合素及Kl9均呈阳性表达。结论:运用Ⅳ型胶原粘附结合K-SFM培养可以实现人表皮干细胞的体外快速分离和培养。

Objective To explore a method for isolation and culture of human epidermal stem cells.Methods The Epidermal stem cell were isolated by adhering to collagen type IV after obtained by digesting human foreskin with Dispase Ⅱ and Tryp and culture in vitro in K-SFM.The expressions of β1-integrin and keratin 19（K19）in epidermal stem cell were detected with immunocytochemical methods,and the colony forming efficiency was also studied .Keratinocytes were served also detected.Results It was revealed by histological observation that colonieswere formed 24 hours after inoculation.The isolated and cultured cell cloning efficiency was higher than that of the control group.Positive expression of β1-integrin and K19 of cultured cells was detected by immunocytochemistry.Conclusion Adult epidermal stem cells could be successfully isolated and cultured by adhension with type Ⅳ collagen and culture with K-SFM