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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 establish a lentiviral system to overexpress stable AKT1 gene in bone marrow mesenchymal stem cells. Methods AKT1 cDNA gene was amplified and AKT1-cDNA expression vector was constructed. Recombinant human AKT1 cDNA was transfected into human embryonic kidney 293T cells with shuttle plasmid pCDH1-AKT1. After amplification, the recombinant plasmid was transfected with the lentiviral vector Pig bone marrow mesenchymal stem cells, observe the fluorescence expression of AKT1 protein and mRNA expression levels by Western blot and RT-PCR. Results The results of double enzyme digestion and sequencing showed that the obtained cDNA was a protein coding for the AKT1 gene. 293T cells and bone marrow mesenchymal stem cells transfected efficiency of more than 90%. The results of Western blot and RT-PCR showed that the AKT1 protein and mRNA expression in stable cells were significantly higher than those in primary cells. Conclusion AKT1-cDNA expression vector was successfully constructed. By using lentivirus system, pig bone marrow mesenchymal stem cells can stably overexpress AKT1 over a long period of time.