抗氧化剂和低氧诱导醌氧化还原酶1基因的表达及其机制  

Expression of human NAD(P)H:quinone oxidoreductase 1 gene induced byantioxidants and hypoxia and the mechanism

作　　者：薛丽君[1] 金中初[1] 夏小俊[1] 陈维亚[2] 李红娟[2] 薛旦[1] 梅汝焕[1] 

机构地区：[1]浙江大学医学院,浙江杭州310031 [2]杭州师范学院医学院

出　　处：《毒理学杂志》2005年第2期92-95,共4页Journal of Toxicology

基　　金：国家自然科学基金资助项目 (39970 649);浙江省自然科学基金项目 (30 1 52 9)

摘　　要：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 whether antioxidants and hypoxia can induce the expression of NAD(P)H:quinone oxidoreductase 1 (NQO_1) gene,inhibit the proliferation of human hepatoma cells SMMC-7721 and the relationship between them;whether antioxidant responsive element(ARE) can mediate gene expression in response to antioxidants and hypoxia.Methods SMMC-7721 human hepatoma cells are exposed to antioxidants and hypoxia,each alone or in combination for 24 h.The enzyme activity was determined by spectrophotometric assay using direct measurement of NQO_1 from cells cultured in microtiter wells.Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) technique was used to measure NQO_1 mRNA levels.Proliferation was estimated using the crystal violet staining technique.Electrophoretic mobility shift assay(EMSA) was employed to assess protein binding to the ARE under all of these conditions.Results Antioxidants potently induce an increase in the activity of NQO_1.p-Tyrosol also caused NQO_1 mRNA expression enhancement in a dose-dependent manner.A statistically significant correlation between NQO_1 mRNA levels and enzyme activities induced by p-tyrosol was evident.Simultaneously,p-tyrosol caused a dose-dependent inhibition of cell proliferation,which well inversely correlated with NQO_1 enzyme activity.p-Tyrosol and BHA induced NQO_1 activity were significantly increased in hypoxia than in normoxia.EMSA of nuclear extracts of SMMC-7721 cells exposed to antioxidants(p-tyrosol and BHA) and hypoxia,or antioxidants combined with hypoxia,showed specific and constitutive protein binding to the wild ARE under all of these conditions.Conclusion Antioxidants p-tyrosol,BHA,β-NF potently induce and increase of NQO_1 activity in hepatoma cells SMMC-7721 under normoxia.Hypoxia further enhances NQO_1 activity induced by p-tyrosol and BHA.In normoxia,enzyme activity of NQO_1 induced by p-tyrosol correlates its mRNA expression increase,and cell proliferation correlates with NQO_1 enzyme activity.ARE is a common regulat

关 键 词：醌氧化还原酶 抗氧化剂 氧诱导 人肝癌细胞SMMC-7721 1基因 NQO1 mRNA表达量 剂量依赖性 RT-PCR 抑制细胞增殖 基因表达及 电泳迁移率 比活力 调节机制 诱导表达 反应元件 EMSA 特异结合 增殖抑制 核蛋白 ARE 低氧 酪醇 

分 类 号：R739.63[医药卫生—肿瘤] R977.9[医药卫生—临床医学]