Oct4介导小鼠原代肝细胞去分化研究

Oct4-mediated dedifferentiation of primary liver cells in mice

目的通过Oct4介导小鼠肝细胞去分化为Sox17阳性细胞,探索肝胰重编程新途径。方法构建慢病毒载体pWPT-Oct4,包装和浓缩病毒;改良两步胶原酶灌流法分离小鼠原代肝细胞;Oct4-慢病毒浓缩液感染小鼠原代肝细胞,RT-PCR检测肝细胞及内胚层转录因子的表达。结果通过酶切、测序鉴定,证实成功构建包含Oct4慢病毒表达载体。感染肝细胞后,外源性Oct4出现表达,成熟的肝细胞转录因子(Alb、Ttr、Afp)表达下降,并表达内胚层早期标记物Sox17,而没有Oct4、Nanog及Sox2等多能性基因的内源性表达。结论 Oct4介导肝细胞去分化,并出现Sox17阳性细胞,为进一步将Sox17阳性细胞分化为胰岛素产生细胞奠定基础。

Objective To study the new approaches to liver and pancreas reprogramming by observing the Oct4-mediated dedifferentiation of liver cells into Sox17 positive cells. Methods A lentiviral vector, pWPT-Oct4, was constructed, and lentivirus was packed and concentrated. Primary liver cells in mice were isolated by the modified two-step collagenase perfusion and infected with the concentrated Oct4 lentivirus fluid. Expression of endoderm and hepatic cell transcription factors was detected by RT-PCR. Results Whether the expression vector containing Oct4 lentivirus was successfully constructed was verified by enzyme digestion and sequencing. Exogenous Oct4 was expressed, the expression level of mature transcription factors in liver cells （Alb, Ttr and Afp） decreased, early endoderm marker Sox17 was expressed while endogenous pluripotency genes, Oct4, Nanog, and Sox2 were not expressed in infected primary liver cells. Conclusion Oct4 mediates dedifferentiation of liver cells in which Sox17 positive cells can be observed, thus laying a foundation for the further differentiation of Sox17 positive cells into insulin-producing cells.