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目的 探讨低氧和抗氧化剂诱导醌氧化还原酶1(NQO1 )基因表达及其与细胞增殖的关系和诱导表达的调节机制。方法 人肝癌细胞SMMC 772 1经抗氧化剂[酪醇(p Ty) ,丁基羟茴香醚(BHA) ,β萘黄酮(βNF) ]、低氧或两者共同处理2 4h ,分别采用微孔板测定法、RT PCR、结晶紫显色法、电泳迁移率变动试验(EMSA)测定MQO1 活力,NQO1 mRNA表达,细胞增殖和细胞核蛋白与抗氧化反应元件(ARE)的结合情况。结果 常氧下BHA ,βNF和p Ty均能诱导NQO1 比活力增加,酪醇诱导NQO1 mRNA表达呈剂量依赖性增加,且与酶比活力存在明显的相关性;酪醇在一定范围内,其抑制细胞增殖的能力呈剂量依赖性,且细胞增殖与酶比活力存在负相关。低氧与抗氧化剂共同处理比常氧下抗氧化剂诱导NQO1 比活力有增加(r=-0 41,P <0 0 1)。EMSA试验表明,ARE能与低氧、抗氧化剂或低氧+抗氧化剂诱导的核蛋白特异结合。结论 抗氧化剂在常氧下可诱导人肝癌细胞NQO1 活力增加,低氧加强其诱导能力。其中酪醇诱导NQO1 活力与NQO1 mRNA表达量增加相关,同时酪醇能抑制细胞的增殖,这种增殖抑制与酶活力诱导增加有关。ARE参与介导抗氧化剂和低氧诱导NQO1 基因表达的调节。
Objective To investigate the relationship between the expression of quinone oxidoreductase 1 (NQO1) gene induced by hypoxia and antioxidants and its regulation of cell proliferation and its regulation mechanism. Methods Human hepatocellular carcinoma cell line SMMC 772 1 was treated with anti-oxidant (p Ty, BHA, βNF), hypoxia or both for 24 hours. The activity of MQO1, the expression of NQO1 mRNA, the cell proliferation and the combination of nuclear protein and antioxidant element (ARE) were measured by the method of RT PCR, crystal violet colorimetry and electrophoretic mobility shift assay (EMSA). Results Under normoxia, BHA, βNF and p Ty both induced the increase of specific activity of NQO1 and the increase of NQO1 mRNA induced by tyramide in a dose-dependent manner. There was a significant correlation between the specific activity of NQO1 and tyrosol. Cell proliferation in a dose-dependent manner, and cell proliferation and enzyme activity is negatively correlated. Hypoxia and antioxidants co-treatment than the normoxia antioxidant-induced NQO1 specific activity increased (r = -041, P <0.01). EMSA tests showed that AREs specifically bound to hypoxia, antioxidants or hypoxia + antioxidant-induced nuclear proteins. Conclusion Antioxidants can induce the increase of NQO1 activity in human hepatocarcinoma cells under normoxia and hypoxia to enhance its induction ability. Tyrosol induced NQO1 activity and NQO1 mRNA expression increased, while tyrosol inhibited cell proliferation, inhibition of this proliferation and increased enzyme activity. ARE is involved in the mediation of antioxidant and hypoxia-induced regulation of NQO1 gene expression.