Oct4介导小鼠原代肝细胞直接重编程为Pdx-1阳性细胞

The Direct Oct4-mediated Primary Liver cells in Mice Reprogramming of Pdx-1 Positive Cells

目的:Oct4介导小鼠原代肝细胞直接重编程为Pdx-1阳性细胞的研究。方法:利用小鼠慢病毒载体pWPT-Oct4联合包装质粒包装病毒,含有PEG6000的病毒浓缩液对病毒原液进行浓缩;慢病毒浓缩液感染利用两步胶原酶灌流法分离培养的小鼠肝细胞;RT-PCR检测肝脏、胰腺谱系及多能性转录因子表达。结果:含有Oct4慢病毒感染小鼠原代肝细胞后,Oct4出现表达,肝细胞相关转录因子(Alb、Afp)表达逐渐下降,内胚层早期标志物Foxa2的表达随时间的增加而减弱,Sox17在感染后12d、18d出现表达,并表达胰腺发育过程中的关键性基因-胰十二指肠同源异盒蛋白-1(pancreatic duodenal homeobox-1,Pdx-1);不表达Nanog及Sox2等多能性基因。结论:在Oct4介导下成功去分化小鼠原代肝细胞,出现Pdx-1阳性细胞。

Objective： To explore new ways of liver and pancreas reprogramming through the experiment that Oct4-mediated mouse liver cells differentiate the Pdx-1 positive cells. Methods： The lentivirus was packed and concentrated by lentiviral vector pWPT-Oct4, followed by the isolation of mouse primary hepatocytes by the modified two-step collagenase perfusion and then they were infected by the Oct4 lentivirus. Expressions of the pancreas transcription factors and the hepatocytes transcription factors were detected by RT-PCR. Results： Expression of exogenous Oct4 appeared, and expressions of mature transcription factors in liver cell （Alb, Ttr and Afp） decreased, then expression of early endoderm markers Pdx-1 appeared in the infected mouse primary hepatocytes and the expression of endogenous pluripotency genes Nanog, and Sox2 did not appear. Conclusion： Pdx-1 positive cells appear in Oct4-mediated hepatocyte, which can provide a foundation for the fiarther differentiation into insulin-producing cells.