利用诱导因子Oct4、Sox2和SV40T的mRNA介导体细胞重编程研究

Cellular Reprogramming by in vitro Transcribed mRNA Cocktail of Oct4,Sox2 and SV40 T-antigen

为利用特定诱导因子Oct4、Sox2和SV40T的体外转录mRNA实现安全的成纤维细胞重编程,本研究成功构建了特定诱导因子Oct4、Sox2和SV40T的mRNA体外转录载体,并对体外转录mRNA进行了5′和3′端加工修饰;进行了诱导因子mRNA的293和IMR90细胞转染试验,利用免疫细胞化学、免疫荧光和Real-Time PCR分别检测了Oct4、Sox2、SV40T和Nanog的表达情况。结果显示,以上2种细胞以Oct4∶Sox2∶SV40T=2∶1∶1的mRNA比例转染后均表达这3种特定诱导因子,且相应蛋白都正确地定位在细胞核上。转染细胞中Oct4和Nanog的表达都特异性地增高。结果提示,3种诱导因子的体外转录mRNA能够在体内协同作用,激活细胞内源性干性标志基因Nanog的表达,为开启重编程过程奠定了坚实的基础。

The aim of this study was to reprogram fibroblasts cell by in vitro transcribed mRNA cocktail of Oct4, Sox2 and SV40 T-antigen, and provide a safe, nonintegrating strategy for somatic cell reprogramming. The reeombinated mRNA transcription vectors including the above three induced facotrs genes were constructed and transcripted, respectively, then 5IUTR and 3I UTR of β-globin gene were used for stabilizing in vitro transcribed mRNA following in vitro cap- ping and poly（A） tailing. After mRNA transfection of 293 and IMRg0 cells, immunocytochemistry, immunoflurescence and Real-Time PCR methods were used to detect gene expression, pro- tein location and endogenous gene expression related to pluripotence. The results showed that target gene expressions could be detected in 293 and IMR90 cells and all expressed protein were localized properly in the nucleus. The specific expression of Oct4 and Nanog were increased in transfection cells. Endogenous Nanog expression was induced by mRNA cock-tail transfection. These findings indicate that mRNA of Oct4, Sox2 and SV40 T in vitro can coorperate to initiate cellular reprogramming.