人表皮干细胞的体外分离与培养

目的 探索人表皮干细胞（epidermal stem cells，ESCs）的分离方法和培养体系。方法 用Ⅳ型胶原纯化、富集ESCs，将黏附细胞（实验组）和未黏附细胞（对照组）分别接种在Ⅳ型胶原摹质（实验组为A1，对照组为A2）和3T3细胞滋养层（实验照组为B1，对照组为B2），培养体系为：低糖无钙DMEM培养基（添加10％胎牛血清、表皮生长因子10μg／L、氯化钙0．05mmol／L、氢化可的松0．8mg／L），观察细胞能否呈克隆状生长，用流式细胞仪和免疫细胞化学染色，对ECSs周期和表型进行分析。结果 实验组细胞呈克隆状生长，G0／G1期细胞和α6^briCD71 dim细胞百分率明显高于对照组，差异有统计学意义（P〈0．05），实验组角蛋白19免疫细胞化学染色呈阳性，对照组呈刚性。结论 人ESCs可通过Ⅳ型胶原快速黏附分选，并可在适当的培养体系里扩增。

SEPARATION AND CULTURE OF HUMAN EPIDERMAL STEM CELLS IN VITRO

OBJECTIVE: To investigate the culture method for epidermal stem cells in vitro. METHODS: The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37 C to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on collagen IV or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetal bovine serum, 10 microg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0. 8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase constrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. RESULTS: The cells selected by rapid adherence to collagen IV formed large colonies at 7- 8 days, expressed K19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of alpha 6 briCD 71 dim cells in the experimental groups were higher than those in the control group. It indiciated that there was a significant difference between the experimental groups and the control groups(P<0. 05). CONCLUSION: The human epidermal stem cells can be selected by rapid attachment to collagen IV, and they can be expanded in culture if the appropriate conditions are maintained.