修复基因MTH1与OGG1在H_2O_2诱导的DNA氧化性损伤中的作用研究

Effects of MTH1 and OGG1 in Repair of DNA Oxidative Damage Induced by H_2O_2

背景与目的:利用H2O2作用于Ⅱ型人肺泡上皮细胞A549,分析8-羟基脱氧鸟苷(8-oxo-dG)的形成,探讨修复基因hMTH1与hOGG1在DNA氧化性损伤中的作用。材料与方法:在A549细胞培养液中分别加入终浓度为0、50、100、200和300μmol/L的H2O2孵育不同时间(0、6、12、18和24h)后,利用高压液相色谱串联电化学检测技术、核酸内切酶切割法及RT-PCR技术分析细胞8-oxo-dG水平、8-oxo-dG修复酶活性及hOGG1和hMTH1基因表达水平。结果:与对照组比较,100、200、300μmol/LH2O2浓度时均能使A549细胞DNA的8-oxo-dG水平增高(P<0.05或P<0.01)。200μmol/LH2O2作用12h后8-oxo-dG水平上升到峰值并在24h降至基线水平,8-oxo-dG水平在6～18h处理组显著高于对照组(P<0.05或P<0.01)。而8-oxo-dG修复酶活性在200μmol/LH2O2暴露后12～24h均高于对照组(P<0.05或P<0.01),在18h达到峰值。hOGG1及hMTH1mRNA的表达水平也因H2O2暴露而升高(P<0.05或P<0.01),但峰值在时间上交替出现。结论:H2O2能诱导8-oxo-dG修复酶活性增高,hOGG1及hMTH1在DNA氧化损伤的修复活动中呈现时间上的交替关系。

BACKGROUND AND AIM：To assess the interactions of MutT homologue（MTH1） and 8-oxoguanine-DNA glycosylase1（OGG1）,two important genes of base excision repair.We examined 8-hydroxyguanine（8-oxo-dG） formation and 8-oxo-dG repair enzyme activity in pulmonary type-II-like epithelial cells to determine the association of hOGG1 and hMTH1 under oxidative stress induced by H2O2.MATERIALS AND METHODS：A549 cells were incubated with H2O2 at concentrations of 0,50,100,200 and 300 μmol/L for 24 h.We then evaluated 8-oxo-dG formation, 8-oxo-dG repair enzyme activity and expressions of hOGG1 and hMTH1 genes. This was done using a high-performance liquid chromatography system equipped with an electrochemical detector, endonuclease nicking assay and reverse transcription polymerase chain reaction, respectively. RESULTS： H2O2 induced the formation of 8-oxo-dG in DNA at concentrations of 100 and 200 Vmol/ L. 8-oxo-dG levels peaked at 12 h and had declined to near baseline at 24 h, whereas 8-oxo-dG repair enzyme activity peaked at 18 h post-H2O2 exposure, hOGG1 and hMTH1 mRNA levels were also increased by H2O2 exposure, with alternating peaks. CONCLUSION： These data suggested that hOGG1 and hMTH1 displayed alternating effects in the process of oxidized DNA repair.