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目的 探讨过氧化氢 (H2 O2 )对肠上皮细胞线粒体DNA(mtDNA)结构与功能的损伤作用。方法 肠上皮细胞株 (SW 480 ) ,采用 4mmol LH2 O2 处理细胞后进行线粒体DNA的提取 ,对细胞色素C氧化酶 (COXⅠ、COXⅡ、COXⅢ )基因进行聚合酶链反应 (PCR )扩增 ,并将PCR产物进行直接测序 ;同时于该时相点测定酶活性。结果 细胞色素C氧化酶COXⅠ从 6 80 3～ 6 848出现大范围的点突变及 70 2 7出现点突变 (TC) ,在 732 9(CG)、7341(TG)、735 2 (GA)出现点突变 ,在7337出现缺失突变 (缺T) ;COXⅡ序列在 82 19～ 82 6 0段出现大范围的点突变 ;H2 O2 可导致mtD NA突变基因所编码的细胞色素C氧化酶活性明显下降。结论 H2 O2 可明显损伤mtDNA编码的细胞色素C氧化酶基因 ,这种损伤作用最终导致其编码的酶蛋白活性明显下降。
Objective To investigate the effect of hydrogen peroxide (H2O2) on the structure and function of mitochondrial DNA (mtDNA) in intestinal epithelial cells. Methods Intestinal epithelial cell line (SW 480) was treated with 4mmol LH2 O2 and the mitochondrial DNA was extracted. The cytochrome C oxidase (COXⅠ, COXⅡ, COXⅢ) gene was amplified by polymerase chain reaction (PCR) PCR products were sequenced directly; meanwhile enzyme activity was measured at this time point. RESULTS: A wide range of point mutations in COXⅠ and a point mutation in 70 2 7 were found at 6 803 ~ 6 848. There was a significant difference between 7329 (CG), 7341 (TG) and 735 2 (GA) Mutation, a deletion mutation (lack of T) was found in 7337; a large range of point mutation was found in COXⅡ sequence in 8219 ~ 8260; H2O2 could lead to a significant decrease of cytochrome C oxidase activity encoded by mtD NA mutant gene. Conclusion H2O2 can obviously damage cytochrome C oxidase gene encoded by mtDNA, which results in a marked decrease of the activity of enzyme protein encoded by H2O2.