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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: Oct4-mediated direct reprogramming of mouse primary hepatocytes into Pdx-1 positive cells. Methods: The viral lentivirus was encapsulated by using the lentiviral vector pWPT-Oct4 and the virus concentrate containing PEG6000 was concentrated. The lentiviral concentrated solution was used to isolate the cultured mouse hepatocytes by two-step collagenase perfusion method. RT -PCR detection of liver, pancreatic lineage and pluripotency transcription factor expression. Results: The expression of Oct4, the expression of hepatocyte related transcription factor (Alb, Afp) and the expression of Foxa2, an early marker of endoderm, were weakened with the passage of time. 12d and 18d after infection, the expression of pancreatic duodenal homeobox-1 (pancreatic duodenal homeobox-1), a key gene in the process of pancreatic development, was not expressed Energy gene. Conclusion: Pdx-1 positive cells appeared in primary dendritic cells induced by Oct4.