胎鼠表皮干细胞的分离培养及毛囊再生研究

目的：分离培养胎鼠的表皮干细胞及毛囊乳头层细胞，通过动物模型观察两者复合移植后，上皮形成及毛囊的再生情况。方法：采用IV型胶原分离培养胎鼠表皮干细胞，检测β1整合素及角蛋白15的表达水平，测定其细胞周期及克隆形成率（以角质细胞为对照）；分离培养毛囊乳头层细胞，并接种在纤维蛋白胶中，与表皮干细胞膜片复合形成全层皮肤等同物，移植在裸鼠全层皮肤缺损创面作为实验组，同时将胎鼠成纤维细胞替代乳头层细胞作为对照组，8－10周后观察创面及囊的组织学变化。结果：胎鼠表皮干细胞表达高水平的β1整合素及角蛋白15；细胞周期分析显示，G1期细胞百分率为94．9％，S期为3．5％，提示为慢循环细胞周期；角质细胞的G1期细胞有百分率为74．1％，S期为17．5％；表皮干细胞的克隆形成率为15．3％，明显高于对照组的6．7％；裸鼠创面愈合后8－10周，实验组可见新生毛囊形成，对照组未见到此现象。结论：能初步实现对胎鼠表皮干细胞的体外分离培养；在毛囊乳头层细胞的诱导下，胎鼠表皮干细胞可参与形成新的毛囊结构。

In vitro culture of murine fetal epidermal stem cell and its relationship with the regeneration of hair follicle

OBJECTIVE: To isolate and culture the murine fetal epidermal stem cells (ESCs) and folliculus pili cells (FPCs) in vitro, and to observe the regeneration of hair follicle and epidermis after cografting of ESCs and FPCs. METHODS: The ESCs were isolated by adhering to murine type IV collagen and were cultured in conditional medium. The expression level of beta1-integrin and keratin 15 in ESCs was detected. At the same time, the cell cycle and clony forming eficiency (CFE) in ESCs were also determined. The FPCs were isolated and cultured and inoculated in fibrin-gel to form FPCs-gel. A full skin equivalent was prepared by combining ESCs with FPCs-gel and was grafted onto total skin loss wounds on the back of BALB/C nude mice. The histological changes of the wounds and the hair follicles were observed at 8 - 10 weeks after the grafting. RESULTS: There were high level expressions of beta1-integrin and keratin 15 in murine fetal ESCs. It was indicated by cell cycle analysis that cells in G1 stage accounted for 94.9% of the cells, while that in S stage, 3.5%, suggesting slow cell cycle. Nevertheless, the keratinocytes in G1 stage accounted for 74.1% and that in S stage, 17.5% of cells in control group. The CFE of ESCs was 15.3%, and it was much higher than that in control group (6.7%). The newly formed hair follicles could be found in the grafted rats but not in the control group 8 - 10 weeks after the wound healing in nude rats. CONCLUSION: The ESCs could be successfully isolated and cultured in vitro and might participate in the formation of hair follicle structure under the induction of FPCs.