牛表皮祖细胞的体外分离培养

目的]探索和建立牛表皮祖细胞体外分离与培养体系。方法]无菌取3个月龄的胎牛皮肤组织，用组织块法培养获得细胞，用L—DMEM培养基（添加5％FBS，0．05mmol／LCaCl2，20ng／mlEGF，20ng／mlbFGF，1％B-27，0．4斗g／mlhydrocortisone）置细胞培养箱内培养。在显微镜下观察细胞的克隆形成能力，免疫细胞化学染色鉴定a6、B1整合素。结果]获得的细胞有很高的克隆形成能力，a6、p1整合素免疫细胞化学染色阳性。RT—PCR检测结果表明，P，代时Oct4基因有表达，但传代超过P11代时，Oct4基本不表达。结论]该研究可为进一步采用组织工程技术构建皮肤组织奠定基础。

Separation and Culture of Epidermal Progenitor Cells in vitro

Objective] The research aimed to investigate and establish the culture method for epidermal progenitor cell in vitro.Method] The calf embryos′skin about three months was peeled from the subcutaneous tissue gently under sterile conditions.The L-DMEM culture medium supplemented with 0.05 mmol/L CaCl2,5% FBS,20 ng/ml EGF,20 ng/ml bEGF,1% B-27 and 0.4 μg/ml hydrocortisone.Cultures were observed for colony formation under a phase contrast microscope.Meanwhile,the expression of α6 integrin and β1 integrin was also detected by immunohistochemistry staining.Result] The results showed that the epidermal progenitor cells had higher colony for ming efficiency,and α6 integrin and β 1integrin were strongly expressed in the cultured epidermal stem cells.The expression of Oct4 in P3 and P11 cells detected by RT-PCR showed that the epidermal stem cells in P3 had high expression of Oct4,but P11 had little expression.Conclusion] The study can lay basis for establishing the skin tissue using tissue-engineered technique in future.