【摘要】 目的探索人表皮干细胞(ep iderm a l stem ce lls,ESC s)的分离方法和培养体系。方法用Ⅳ型胶原纯化、富集ESC s,将黏附细胞(实验组)和未黏附细胞(对照组)分别接种在Ⅳ型胶原基质(实验组为A1,对照组为A2)和3T 3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DM EM培养基(添加10%胎牛血清、表皮生长因子10μg/L、氯化钙0.05 mm o l/L、氢化可的松0.8 m g/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α6briCD71d im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,对照组呈阴性。结论人ESC s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。更多还原

【Abstract】 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℃ 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 μg/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 α₆^briCD₇₁^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.更多还原