锰超氧化物歧化酶及其C47T变异对耳蜗毛细胞氧化损伤作用的影响

目的 探讨锰超氧化物歧化酶(SOD2)及其C47T变异在耳蜗毛细胞氧化损伤中的作用.方法 用Ala16型(突变克隆)和Val16型(野生克隆)SOD2转染HEI-OC1细胞,同时设立未转染和空质粒转染对照.噻唑兰(MTr)法检测细胞的增殖情况;黄嘌呤氧化法检测胞内SOD2活力.100 μmol/L叔丁基过氧化氢(t-BHP)对其染毒12 h后,2'-7'-二氯荧光黄双乙酯(DCFH-DA)法检测胞内活性氧(ROS)水平;Annexin V/PI双标记后通过流式细胞仪检测细胞早期凋亡率和坏死率或晚期凋亡率.结果 SOD2表达质粒和空质粒转染不影响HEI-OC1细胞的增殖;Ala16SOD2和Val16 SOD2转染组SOD2活力分别是转染对照组的3.51和3.71倍,差异有统计学意义(P＜0.01),2种不同基因型间SOD2活力的差异无统计学意义(P＞0.05).染毒后,DCFH-DA方法 显示,未转染和空质粒对照组绝大部分细胞均发出++级明亮荧光,Ala16型和Val16型SOD2转染组中均只有约50%细胞发出±～+级别的模糊荧光.HEIOC1细胞早期凋亡率和坏死率或晚期凋亡率均下降,差异有统计学意义(P＜0.01),但是,其在SOD2 2种基因型之间的差异无统计学意义(P＞0.05).结论 SOD2 3.71倍以下高水平的表达可以降低氧化应激耳蜗毛细胞胞内ROS水平,而SOD2 C47T变异则对之无影响.SOD2为NIHL易感基因,rs4880多态性位点则与NIHL遗传易感性无直接功能性的相关.

Abstract：

Objective To study the effect of SOD2 and its C47T mutation on oxidative injury in cochlea hair cells. Methods HEI-OC1 cells were transfected with the SOD2 of Ala16 and Vla16. Cells' proliferation ability was determined by MTT assay. The intracellular SOD2 activities were detected by xanthine oxidase method. Intracellular ROS were determined by DCFH-DA after exposure to 100 μ mol/L t-BHP and the early apoptotic and necrotic rate or late apoptotic rate were quantified by flow cytometry (FCM) using Annexin V/PI double staining. Results MTT method showed the transfection of SOD2 gene and empty plasmid did not affect the proliferation capacity. SOD2 vitality in Ala16 and Val16 SOD2 transfected cells increased 2.51 and 2.71 times respectively (P＜0.01), but the difference between the two transfection groups was not statistically significant (P＞0.05). After exposed to t-BHP, the majority of the untransfected and empty plasmid transfected cells sent '++' class bright fluorescence, while in Ala16 and Val16 SOD2 transfected groups, only about half cells sent '± '～'+' level fuzzy fluorescence, determination of FCM suggested the early apoptotic and necrotic rate or late apoptotic rate decreased after SOD2 transfection (P＜0.01), but the difference between the two genotypes of SOD2 was not statistically significant (P＞0.05). Conclusion High expression of SOD2 below 3.71 times can reduce intracellular ROS level in HEI-OC1 cells, while SOD2 C47T mutation had no effect on them. SOD2 can be considered as NIHL susceptibility gene and its rs4880 SNP may be not directly related to NIHL genetic susceptibility.

Effects of SOD2 and its C47T mutation on oxidative injury in cochlea hair cell

Objective To study the effect of SOD2 and its C47T mutation on oxidative injury in cochlea hair cells. Methods HEI-OC1 cells were transfected with the SOD2 of Ala16 and Vla16. Cells' proliferation ability was determined by MTT assay. The intracellular SOD2 activities were detected by xanthine oxidase method. Intracellular ROS were determined by DCFH-DA after exposure to 100 μ mol/L t-BHP and the early apoptotic and necrotic rate or late apoptotic rate were quantified by flow cytometry (FCM) using Annexin V/PI double staining. Results MTT method showed the transfection of SOD2 gene and empty plasmid did not affect the proliferation capacity. SOD2 vitality in Ala16 and Val16 SOD2 transfected cells increased 2.51 and 2.71 times respectively (P＜0.01), but the difference between the two transfection groups was not statistically significant (P＞0.05). After exposed to t-BHP, the majority of the untransfected and empty plasmid transfected cells sent '++' class bright fluorescence, while in Ala16 and Val16 SOD2 transfected groups, only about half cells sent '± '～'+' level fuzzy fluorescence, determination of FCM suggested the early apoptotic and necrotic rate or late apoptotic rate decreased after SOD2 transfection (P＜0.01), but the difference between the two genotypes of SOD2 was not statistically significant (P＞0.05). Conclusion High expression of SOD2 below 3.71 times can reduce intracellular ROS level in HEI-OC1 cells, while SOD2 C47T mutation had no effect on them. SOD2 can be considered as NIHL susceptibility gene and its rs4880 SNP may be not directly related to NIHL genetic susceptibility.