人皮肤成纤维细胞来源诱导性多潜能干细胞的建立和鉴定

Generation of human induced pluripotent stem cells from human skin fibroblasts

目的建立和鉴定人皮肤成纤维细胞（HSF）来源的诱导性多潜能干细胞（iPS）。方法利用逆转录病毒将Sox2、Klf4、Oct4、c-Myc基因导入原代HSF细胞，将其编程为iPS细胞。检测细胞内源、外源基因表达量，鉴定外源基因是否插入iPS细胞，分析细胞核型，细胞碱性磷酸酶染色和免疫荧光染色，体内分化畸胎瘤，体外分化拟胚体实验，对建立的iPS细胞进行鉴定。结果（1）iPS细胞形态类似于胚胎干细胞（ES）。（2）iPS细胞内源多能基因（Nanog、Oct4、Rexl、Sox2）表达量增高，外源编程基因（Sox2、Klf4、Oct4、c-Myc）表达量降低。（3）琼脂糖凝胶电泳实验证实外源基因插入iPS细胞核内。（4）iPS细胞核型正常，碱性磷酸酶活性增高，表达ES细胞特异性蛋白。（5）iPS细胞在体内能分化为畸胎瘤，在体外能分化为拟胚体。结论新建立的iPS细胞类似于ES细胞具有多向分化潜能。

Objective Generation of human induced pluripotent stem cells from human skin fibro- blasts. Methods Sox2, Klf4, Oct4, c-Myc were transfected into HSF cells with retrovirus, and then HSF cells was reprogrammed to iPS cells. Detecting cells endogenous and exogenous gene, analyzing karyotype, cells alkaline phosphatase staining and immunofluorescence staining, differentiating into teratomas in vivo and embryoid bodies in vitro were performed. Results iPS cell morphology was similar to embryonic stem cells （ES）. The expression of Nanog, Oct4, Rexl, Sox2 in iPS cells were higher than HSF cells, and Sox2, Klf4, Oct4, c-Myc were silenced for the iPS cells. Exogenous genes were inserted into the nucleus of iPS cells, which was confirmed by 1% agarose gel electrophoresis, iPS cell karyotype was normal, alkaline phosphatase activity increased, ES cell-specific protein expressed, iPS cells were differentiated into a teratoma in vivo and embryoid bodies in vitro. Conclusions iPS cells were similar to ES cells, which have plunpotency