重组2型腺相关病毒介导锰超氧化物歧化酶转染大鼠耳蜗血管纹缘细胞的研究

Recombinant adeno-associated virus serotypes 2 mediated delivery and expression of MnSOD in cultured strial marginal cells

目的探讨以重组腺相关病毒(Recombinant adeno-associated virus of the serotypes2,rAAV2)为载体对体外培养的大鼠耳蜗血管纹缘细胞(Marginal cells,MCs)转染锰超氧化物歧化酶(Manganese SuperoxideDismutase,MnSOD),获得高表达MnSOD转基因缘细胞的可行性。方法实验组按感染复数(MOI)为104v.g./cell对体外培养的血管纹缘细胞转染rAAV2-MnSOD-EGFP,同时设转染rAAV2-EGFP缘细胞作为空载对照组,未转染细胞为空白对照组。荧光显微镜下每2d观察1次MCs的绿色荧光蛋白(Enhenced green fluorescentprotein,EGFP)表达情况。比色法测定各组MCs的MnSOD活性。激光共聚焦显微镜下观察转染rAAV2-MnSOD-EGFP后MnSOD的表达,免疫印迹法(Western blot)定量分析MnSOD的表达。结果(1)各组MCs转染后,生长正常,空载对照组MCs转染rAAV2-EGFP后2天开始出现微弱EGFP的表达,1周后至高峰;实验组转染rAAV2-MnSOD-EGFP后4d出现EGFP的表达,10d至高峰,且持续表达于细胞培养的整个期间。EGFP的表达在荧光显微镜490nm波长激发光下呈黄绿色,弥漫于整个胞质。(2)对激光共聚焦显微镜及Westernblot的检测结果进行分析,实验组较空载对照组和空白对照组的MCs中MnSOD的表达明显升高,差异有统计学意义。结论rAAV2-MnSOD-EGFP能有效地转染体外培养的MCs并持续高表达MnSOD。

Objective To obtain transgenic marginal cells （MCs） of the rat cochlea stria vascularis by recombinant adeno-associated virus serotypes 2 （AAV2） mediated delivery of cDNA of manganese superoxide dismutase （MnSOD）. Methods Cultured rat MCs were infected with rAAV2-MnSOD-EGFP at dosage of multiplicity of infection （MOI） 10^4 v.g./cell and with rAAV2-EGFP as control. The expression of EGFP in MCs was examined using a fluorescence microscope every 2 days after infection. The activity of MnSOD was determined by colorimetric assays. The expression of MnSOD in MCs was examined using western blot and laser scanning confocal microscopy. Results （1）The expression of EGFP in MCs could not be detected until 2 days after rAAV2-EGFP infection and until 4 days after rAAV2-MnSOD-EGFP infection. The positive expression of EGFP in MCs reached fastigium after a week and lasted over a month. （2） According to the results of western blot test and the laser scanning confocal microscopic examination, the concentration of MnSOD in the MCs after rAAV2-MnSOD-EGFP infection was higher than that in the control cells. Conclusion The rAAV2-MnSOD-EGFP can effectively transfect cultured MCs, and the transgenic cells show a high expression of MnSOD.