重组2型腺相关病毒介导锰超氧化物歧化酶高表达对耳蜗血管纹缘细胞氧化损伤的保护作用

The recombinant adeno-associated viruses of the serotypes 2 mediated overexpression of MnSOD protects against oxidation-induced apoptosis in stria marginal cells of rat cochlea

目的:探讨以重组2型腺相关病毒(AAV2)介导锰超氧化物歧化酶(MnSOD)高表达对耳蜗血管纹缘细胞氧化损伤的作用及机制。方法:体外培养耳蜗血管纹缘细胞(MCs),分为实验组、对照组及正常组。实验组按感染复数(MOI)为104v.g/cell感染rAAV2-MnSOD-EGFP,同时感染rAAV2-EGFP作对照组及不作处理缘细胞为正常组。荧光显微镜下观察MCs绿色荧光蛋白(EGFP)的表达情况及免疫印迹法(western blot)分析转染后MnSOD的表达水平。实验组及对照组同时暴露于400μmol/L H2O22 h,24 h后比色法测定各组丙二醛(MDA)含量;流式细胞PI荧光标记检测细胞凋亡率;Western blot检测凋亡因子Caspase-3活化片段Cleavedcaspase-3的表达。结果:①各组MCs感染病毒后,生长正常,实验组感染rAAV2-MnSOD-EGFP后4d出现EG-FP的表达,10 d至高峰;且持续细胞体外生长的整个周期。实验组MnSOD的表达及活性明显高与对照组及正常组,差异有统计学意义(P<0.05)。②经H2O2作用后实验组MDA、Caspase-3活化片段Cleaved caspase-3的表达及凋亡率明显低与对照组,差异有统计学意义(P<0.05)。结论:rAAV2-MnSOD-EGFP能有效地转染体外培养的MCs使其高表达MnSOD,并对MCs氧化损伤有保护作用,这种保护作用与抑制凋亡因子Caspase-3的活化有关。

Objective：To investigate the influence of overexpression of manganese superoxide dismutase（MnSOD）of stria marginal cells（MCs） of the rat cochlea by the recombinant adeno-associated viruses of the serotypes 2（AAV2） mediated gene-delivery for hydrogen peroxide-induced oxidative stress in vitro.Method：Primary cultures of MCs were infected using rAAV2-MnSOD-EGFP at dosage of multiplicity of infection（MOI） 104v·/cell and using rAAV2-EGFP as control.The expression of MnSOD in MCs was examined using western blot and the activity of MnSOD was determinated by colorimetric assays.Oxidative stress was induced in MCs by exposing them to H2O2（400 μmol/L） for 2 hour and reculturing them in normal medium.After 24 h the amount of the lipid peroxidation production malondialdehyde（MDA）was detected.Apoptosis was assessed by flow cytometry by Propidium iodium staining.The expression of the cleaved Caspase-3 was assessed by Western blot.Result：①EGFP expression in MCs could not be detected until 4 days after rAAV2-MnSOD-EGFP infection and reached fastigium after 10 days and lasted over a month.The MnSOD level in the rAAV2-MnSOD-EGFP group was higher than that in the control group.②After being exposed to H2O2,the amounts of MDA in rAAV2-MnSOD-EGFP group,control group and normal group were 0.464±0.049,1.103±0.033 and 0.185±0.005（nmol/mg prot）,respectively.The expression of the cleaved-caspase-3 in rAAV2-MnSOD-EGFP group was lower than that in control group and the number of apoptotic cells decreased significantly.Conclusion：The results demonstrate that the rAAV2-MnSOD-EGFP can effectively transfect cultured MCs,and the transgenic cells show a high expression of MnSOD which can proctect the MCs against oxidative challenge.The role of overexpression MnSOD in MCs apoptosis induced by oxidative injury may be associated with suppressing the activation of caspase-3.