目的 研究氧化应激对klotho基因启动子活性的影响及抗氧化系统的作用.方法 构建klotho基因启动子片段的荧光素酶报告基因重组质粒,将重组质粒和内对照质粒海肾荧光素酶报告质粒以脂质体法共转染Hela细胞,以0、400、600、800和1000μmol/L的过氧化氢处理Hela细胞,依次分为对照组、1组、2组、3组和4组,观察klotho基因启动子的活性变化,并分析总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)和过氧化氢酶(CAT)等抗氧化酶的活性变化对此过程的影响.结果 与对照组比较,1组的klotho基因活性无明显改变(P＞0.05);2组、3组和4组的klotho基因启动子活性下降(P＜0.01),2组、3组和4组T-AOC、CAT和GSH-PX活性明显降低(P＜0.05,P＜0.01),与2组比较,3组和4组T-AOC和GSH-PX活性明显降低(P＜0.05).klotho 基因启动子活性下降与T-AOC、CAT和GSH-PX活性下降呈正相关(r=0.805、0.812、0.944,P=0.00).结论 氧化应激下调klotho基因的表达,可能与其下调该基因的启动子活性密切相关,且氧化应激过程中抗氧化酶活性的变化与klotho基因启动子活性的下降可能相关.%Objective To investigate the effect of oxidative stress on klotho promoter's activity in Hela cells,and assess the effect of antioxidation system in this process. Methods Klotho promoter reporter gene plasmid was constructed and transfected into Hela cells. The Hela cells were exposed to hydrogen peroxide at concentrations of 0,400,600,800 and 1000 ptmol/L to form control group and groups 1,2,3,4. Then the activity of klotho promoter was observed. At the same time, the total antioxidation capacity (T-AOC) and activities of catalase(CAT) ,glutathion peroxidase (GSH-PX) were detected and their correlation with the activity of klotho promoter was analyzed. Results Compared with control group, the activity of klotho gene in group 1 had no significant change (P > 0. 05),while the activity of klotho promoter in groups 2,3 and 4 was decreased (P< 0. 01), the activity of T-AOC, CAT and GSH-PX in groups 2, 3 and 4 was significantly decreased (P < 0. 05,P < 0. 01). Compared with group 2,the activity of T-AOC and GSH-PX in groups 3 and 4 was significantly decreased (P < 0. 05). The downregulation of klotho promote's activity was positively correlated with derease in activity of T-AOC, CAT and GSH-PX (r = 0. 805,0. 812,0. 944,P = 0. 00). Conclusion Oxidative stress can down-regulate klotho promoter activity,which may be effected by oxidation resistance.
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