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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 explore the new way of hepatorenal reprogramming by means of Oct4-mediated dedifferentiation of mouse hepatocytes into Sox17-positive cells. Methods The lentiviral vector pWPT-Oct4 was constructed and packaged for virus enrichment. Two-step collagenase perfusion method was used to isolate mouse primary hepatocytes. Oct4-lentivirus concentrate was used to infect mouse primary hepatocytes. RT-PCR was used to detect hepatocytes and Endodermal transcription factor expression. The results of digestion, sequencing identification, confirmed the successful construction of Oct4 lentiviral expression vector. After infected with hepatocytes, exogenous Oct4 was expressed, the expression of mature hepatocyte transcription factors (Alb, Ttr, Afp) decreased, and the endodermal early marker Sox17 was expressed without the pluripotency genes Oct4, Nanog and Sox2 Endogenous expression. Conclusion Oct4 mediated hepatocyte dedifferentiation and Sox17 positive cells, which laid the foundation for further differentiation of Sox17 positive cells into insulin-producing cells.