重组9型腺相关病毒转染小鼠骨髓间充质干细胞

背景：重组9型腺相关病毒对心肌细胞具有良好的亲和力,是目前研究基因治疗心肌梗死的理想载体。目的：观察携带增强型绿色荧光蛋白基因的重组9型腺相关病毒（r AAV9-e GFP）对小鼠骨髓间充质干细胞的转染效率。方法：将携带增强型绿色荧光蛋白基因的重组9型腺相关病毒以不同感染复数（1×105,1×106,1×107）转染体外培养的第4代小鼠骨髓间充质干细胞,并在转染后1-7 d连续用荧光倒置显微镜观察骨髓间充质干细胞中增强型绿色荧光蛋白阳性表达情况,寻找最佳感染条件;采用流式细胞仪检测最佳感染复数下携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞的转染效率。结果与结论：转染后第1天,感染复数为1×107组可见增强型绿色荧光蛋白开始表达,转染后第2天,感染复数为1×105、1×106组开始表达;各组增强型绿色荧光蛋白表达强度随感染复数值的增高而增强,同时增强型绿色荧光蛋白的表达强度随着时间延长而逐渐增强;转染后第5天达到高峰,此时感染复数为1×107组转染效率约8%,结果表明携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞转染效率较低。

Recombinant adeno-associated virus type 9 transfection of mouse bone marrow mesenchymal stem cells

BACKGROUND： Recombinant adeno-associated virus type 9 （rAAV9） has a good affinity to myocardialcytes, and it is an ideal carrier for gene therapy of myocardial infarction. OBJECTIVE： To investigate the transfection efficiency of rAAV9-enhanced green fluorescence protein （eGFP） in transfection of mouse bone marrow mesenchymal stem ceils. METHODS： rAAV9-eGFP was transfected into mouse bone marrow mesenchymal stem cells which were subcultured to the fourth generation at different multiplicities of infection （MOI=1×105 1 ×106, 1 ×107）. To find the best condition of transfection, we observed the eGFP expression under the inverted fluorescence microscope at 1-7 days after transfection. Then the transfection efficiency of rAAV9-eGFP under the optimal MOI was determined by flow cytometry. RESULTS AND CONCLUSION： The cells with rAAV9-eGFP transfection at MOI of 1 ×107 began to exhibit eGFP expression at the 1st day after transfection, and the cells transfected at MOI of 1 × 105 and 1 × 105 began to exhibit eGFP expression from the 2nd day after transfection. The eGFP expression increased with the MOI used for trensfection, and the intensity of eGFP expression increased with the time going on. At the 5th day, the eGFP expression reached the peak value, and meanwhile, the transfection efficiency at MOI=1 ×107 was about 8% rAAV9-eGFP cannot transfect mouse bone marrow mesenchymal stem cells effectively.