实时定量RT-PCR方法检测雌二醇诱导原代培养青鳉鱼肝细胞的基因表达  

Gene expression in primary cultures of male medaka (Oryzias latipes) hepatocytes induced by 17β-estradiol with quantitative real-time RT-PCR

作　　者：王琪[1,2] 黄崇[2] 张照斌[2] 胡建英[2] 张仁陟[1] 

机构地区：[1]甘肃农业大学资源与环境学院,兰州730070 [2]北京大学城市与环境学院,北京100871

出　　处：《环境科学学报》2008年第12期2568-2572,共5页Acta Scientiae Circumstantiae

基　　金：国家自然科学基金项目(No. 40632009);国家重点基础研究发展计划(973)项目(No. 2007CB407304)~~

摘　　要：利用原代培养的雄性青鳉鱼肝细胞进行了相同雌二醇(E2)浓度(100nmol·L-1)下的不同时间(2、4、6、8d)和相同暴露时间(4d)下的不同浓度(1、10、100、1000、10000nmol·L-1)的雌二醇(E2)实验,采用实时定量RT-PCR方法对青鳉鱼VTG-I、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达进行了研究.结果表明, VTG-I、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达与E2的暴露浓度呈现很好的剂量-效应关系,且1nmo·lL-1E2暴露就能显著诱导肝细胞内VTG-I、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达.作为生物监测雌激素活性的in vitro方法,实时定量RT-PCR的灵敏度高于其它已经发表的方法.因此,实时定量RT-PCR方法与原代培养青鳉鱼肝细胞相结合的方法在环境雌激素效应的评价上具有很大地应用潜力.A method of applying quantitative real-time RT-PCR to cultures of male medaka hepatocytes was developed to evaluate estrogenic activity. Primary cultures of male medaka （Orrzias latipes） hepatocytes were exposed to 17β-estradiol （E2） at 100 nmol· L^- 1 for 2, 4, 6, 8-day exposure, and 1, 10, 100, 1000 and 10000 nmol· L^-1E2 for 4 days. The VTG-I, VTG-II, CHG-H, CHG-L and ERα gene expressions were determined using quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）. It was found that dose-dependent VTG-I, VTG-II, CHG-H, CHG-L and α gene expressions in hepatoeytes were induced, and obvious expressions were observed even at 1 nmol· L^ - 1 of E2. This method is more sensitive compared with other in vitro methods for evaluating estrogenic activity. Thus, applying quantitative real-time RT-PCR to primary cultures of male medaka hepatocytes has potential application in the evaluation of estrogenic activity of endocrine disrupting chemicals or environmental samples.

关 键 词：实时定量RT-PCR 雌二醇 原代培养肝细胞 卵黄蛋白原 青鳉鱼 

分 类 号：X171.5[环境科学与工程—环境科学]