线粒体DNA氧化损伤热点的形成及其修复速率的比较

本研究旨在探讨线粒体DNA氧化损伤热点的形成是否与该点DNA修复速率慢有关 .BRL 3A细胞暴露于 5 0mmol·L- 1过氧化氢 (H2 O2 ) 30min后 ,分别于 0 ,1,4及 2 4h收获细胞 ,应用连接介导聚合酶链反应分析线粒体DNA氧化损伤热点的修复百分率并与线粒体DNA氧化损伤的总修复百分率比较 ,通过狭缝印迹法 ,比较了各时间点细胞内p5 3/线粒体DNA比率来验证其修复的可靠性 .结果表明 :在 4及2 4h ,热点的修复速率分别为 5 .2 %和 4 2 .1% .而线粒体DNA的总修复百分率分别为 76 .9%和 84 .1% ,后者与前者相比 ,其修复速率快 1～ 14倍左右 .应用狭缝印迹发现各时间点细胞内p5 3/线粒体DNA比率无明显变化 .因此 ,线粒体DNA氧化损伤热点的形成与其修复速率较慢有关 .狭缝印迹结果表明所观测的线粒体DNA的修复是可信的

Comparison of the repair percentage of oxidative damage hotspots with that of overall oxidative damage in mitochondrial DNA

In order to study whether or not the formation of mtDNA oxidative damage hotspots is related to its slow DNA repair, BRL-3A cells were exposed to 50 mmol·L^-1 H₂O₂ for 30 min and then harvested at 0, 1, 4 and 24 h, respectively. The repair percentage of hotspots was analyzed by the method of LMPCR and then compared with that of overall mtDNA oxidative damage. Blot slot assay was also utilized to show the authenticity of mtDNA repair. Our results showed that at 4 and 24 h, the repair percentage of hotspots were 5.2% and 42.1%, but that of overall mtDNA oxidative repair were 76.9% and 84.1%, the repair rate of overall mtDNA oxidative damage was 1－14 folds faster than that of hospots. The results of slot blot assay showed that p53/mtDNA ratio in cellular total DNA at each time point was not significantly different. These results suggest that the formation of mtDNA oxidative damage hotspotsis related to its slow repair rate, and the results of slot blot assay indicate that our repair rate can authentically describe the real conditions.