AKT1基因过表达骨髓间充质干细胞的建立

Construction of bone marrow stromal cells stably overexpressing AKT1 gene

目的使用慢病毒系统建立AKT1基因过表达的稳转骨髓间充质干细胞。方法扩增AKT1cDNA基因,并构建AKT1-cDNA表达载体,再结合穿梭质粒pCDH1-AKT1转染人胚肾293T细胞进行扩增后,使用慢病毒转染系统进行基因重组,并以其为载体转染猪的骨髓间充质干细胞,观察其荧光表达,采用Western blot和RT-PCR分别检测AKT1蛋白和mRNA表达水平。结果双酶切鉴定和测序结果表明,所获取的cDNA为AKT1的蛋白质编码功能区基因。293T细胞和骨髓间充质干细胞的转染效率均达到90%以上。Western blot和RT-PCR结果表明,稳转细胞的AKT1蛋白和mRNA表达水平均明显高于原代细胞。结论成功构建AKT1-cDNA表达载体;通过使用慢病毒系统,猪骨髓间充质干细胞能长期稳定过表达AKT1。

Objective To construct the bone marrow stromal cells （BMSCs） stably overexpressing AKT1 gene by lentivirus system. Methods The coding sequence of AKT1 cDNA was amplified to construct AKTI-cDNA expression vector, which combined with pCDH1-AKT1 shuttling plamid was transfected into human embryonic kidney 293T cells for amplification. The gene was recombinated using lentivirus system and transfected into porcine BMSCs. The fluorescence expressions of 293T cells and BMSCs were observed under microscope,and the expressions of AKT1 protein and mRNA were detected by Western blot and RT-PCR, respectively. Results Double digestion and sequencing confirmed that the obtained coding sequence of AKT1 cDNA was correct. The transfection efficiency of both 293T cells and BMSCs reached over 90%. Western blot and RT-PCR represented that the expressions of AKT1 protein and mRNA in the stably transfected ceils were higher than those in the primary cells. Conclusion AKTI-cDNA expression vector is successfully constructed. AKT1 can be persistently and stably overexpressed in BMSCs using lentivirus transfection system.