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小鼠体细胞可通过转入干细胞转录因子而诱导生成多能干细胞,这种干细胞称为诱导性多能干细胞(iPSC).利用逆转录病毒、慢病毒、腺病毒、质粒载体、多蛋白表达系统、合成性mRNA及microRNA已成功将小鼠和人的体细胞诱导成多能干细胞.piggyBac是转座频率较高的转座子载体,也已作为载体成功诱导出多能干细胞.为了研究以piggyBac载体诱导出的多能干细胞的体内外分化能力,利用携带Oct4,Sox2,Klf4和c-Myc转录因子的piggyBac转座载体进行了小鼠体细胞的转染诱导,结果诱导获得的iPS细胞碱性磷酸酶染色呈阳性;免疫组化染色OCT4,NANOG,SSEA-1为阳性;RT-PCR检测Oct4,Sox2,c-Myc和Klf4呈阳性.同时诱导获得的iPS细胞系在体外自发分化后,免疫组化染色AFP,BRACHYURY和NCAM1呈阳性,表明分化形成了3个胚层的细胞;该细胞系皮下注射免疫缺陷小鼠iPS细胞形成了畸胎瘤.实验发现,获得的干细胞系可与小鼠8-细胞聚合,进入到嵌合胚胎的内细胞团.这些结果表明,由piggyBac载体诱导的小鼠多能干细胞iPS具有干细胞的主要特性,包括体内外的分化能力.
Mouse somatic cells can be induced to generate pluripotent stem cells by transduction into stem cell transcription factors, which are called induced pluripotent stem cells (iPSCs) .Using retroviruses, lentiviruses, adenoviruses, plasmid vectors, polyprotein expression systems , Synthetic mRNA and microRNA have successfully induced mouse and human somatic cells into pluripotent stem cells.piggyBac is a transposon vector with higher transposition frequency and has also successfully induced pluripotent stem cells as a carrier.In order to study the effect of piggyBac In vivo and in vitro differentiation of pluripotent stem cells induced by the vector, transfection of mouse somatic cells was induced using a piggyBac transposable vector carrying Oct4, Sox2, Klf4 and c-Myc transcription factors, and as a result, the resulting iPS cells were induced to be alkaline Phosphatase positive staining, immunohistochemical staining OCT4, NANOG, SSEA-1 positive; RT-PCR detection of Oct4, Sox2, c-Myc and Klf4 positive.IPS cells were induced simultaneously spontaneous differentiation in vitro, Histochemical staining of AFP, BRACHYURY, and NCAM1 was positive, indicating that 3 germ layers differentiated into cells that subcutaneously immunosuppressed iPS cells to form teratomas. It was found experimentally that the obtained stem cell lines 8-cell mouse polymerization fitted into the inner cell mass of embryos. These results indicate that, in mice induced by piggyBac vector pluripotent stem cells iPS main characteristics of stem cells, including the ability to differentiate vitro and in vivo.