人表皮干细胞体外分离培养及鉴定.docVIP

人表皮干细胞体外分离培养及鉴定[摘要]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。 [关键词]表皮干细胞；细胞培养；细胞分离；鉴定 [中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455（2007）10-1343-03 Isolation,culture and identification of hmnan epithelial stem cells LI Dan,LI Shi-rong,CAO Chuan （Department of Cosmetic Surgery,Southwest Hospital,the Third Military Medical University,Chongqing 400038,China） Abstract: ObjectiveTo explore a method for isolation and culture of human epidermal stem cells. MethodsThe Epidermal stem cell were isolated byadhering 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. ConclusionAdult epidermal stem cells could be successfully isolated and cultured by adhension with type Ⅳ collagen and culture with K-SFM Key words：epidermal stem cells;cell culture;cell isolation;identification 皮肤作为人体最大的器官，外层的表皮终身不断自我更新。这种更新由表皮基底层具有再生能力的角质形成细胞分裂、增殖、分化来完成。皮肤角质形成细胞主要包括表皮干细胞(epidermal stem cell，ESC)、短暂扩增细胞(transit Amplifying cell，TA cell)及终末分化细胞(postmitotic differentiating cell,PMD cell)[2]。其中，位于基底部的表皮干细胞持续增殖分化以取代外层终末分化细胞。从而在维持组织结构的更新、细胞内环境的稳定和创伤后创面修复过程中起着至关重要的作用，但由于ESC 数量较少(只占基底细胞总数的1%～10%[1]),缺乏明确的特异性分子标志,体外培养很容易丢失其干细胞生物学特性，因而如何大量获得纯度较高的表皮干细胞即成为目前干细