基于pGL-4载体的雌激素受体报告基因实验系统的建立与验证  

Establishment and validation of a novel estrogen receptor-mediated reporter gene assay based on pGL-4 vectors

作　　者：刘峰[1] 黄小明[2,3,4] 李云[3] 

机构地区：[1]重庆医药高等专科学校预防医学教研室,重庆400030 [2]西南医科大学公共卫生学院营养与食品卫生教研室,四川泸州646000 [3]四川大学华西公共卫生学院(华西第四医院),四川成都610041 [4]四川省宜宾卫生学校预防教研室,四川宜宾644000

出　　处：《现代预防医学》2017年第10期1846-1850,共5页Modern Preventive Medicine

基　　金：国家自然科学基金重点项目(No.81030053)

摘　　要：目的建立和评价一种新的基于pGL-4载体的雌激素受体报告基因实验系统。方法通过瞬时共转染将雌激素受体表达载体、报告基因载体和内参载体转入LLC-MK2细胞,用特定浓度受试物进行染毒并观察其反应。结果 (1)细胞毒性试验显示,除双酚A和壬基酚在100μmol/L时有细胞毒性外,其余受试物在设定浓度未见细胞毒性。(2)雌激素受体α报告基因实验系统最低检测限为1.9×10^(-11)mol/L,雌二醇和己烯雌酚在10^(-8)mol/L分别产生30.7倍和24.1倍对照组的荧光素酶表达,双酚A、壬基酚和染料木黄酮均能诱导该系统荧光素酶的表达,他莫昔芬和染料木黄酮能抑制雌二醇诱导的荧光素酶表达。(3)雌激素受体β报告基因实验系统中,雌二醇和己烯雌酚分别产生对照组14.4倍和5.6倍荧光素酶表达。结论基于pGL-4载体建立的雌激素受体报告基因实验系统有着良好的重复性、灵敏度和特异度,且能检测不同雌激素受体亚型的激动剂和拮抗剂,是一种新的用于内分泌干扰物大通量筛选的报告基因实验系统。Objective To establish and evaluate a novel estrogen receptor-mediated reporter gene assay based on pGL-4 series vectors. Methods Estrogen receptor expession vector, reporter gene expression vector and internal background plasmid were transiently co-transfected into LLC-MK2cells, and the responses of reporter gene assay to variouschemicals were observed after exposure tochemicals ofdifferent concentrations. Results （1）In cytotoxicity test,chemicals tested did not show any cytotoxicity except for 100μmol/L bisphenol A and nonylphenol. （2）The limit of detection of ERa-mediated reporter gene assay was 1.9×10^-11mol/L. The luciferase expression induced by 17β-estradiol and diethylstilbestrol were 30.7-fold and 24.1- fold of the vehicle control, respectively. Bisphenol A, nonylphenol and genistein also induced luciferase expression. However, tamoxifen and genistein inhibited luciferase expression induced by 17β-estradiol.（3）In ERβ-mediated reporter gene assay, the responses of reporter gene assay to 17β-estradiol and diethylstilbestrol were 14.4-fold and 5.6-fold ofthe control, respectively. Conclusion The ER-mediated reporter gene assay based on pGL-4 vectors has excellent repeatability,sensitivity and specificity,and could detect different ER subtypes of agonists or antagonists. It is a novel and successful reporter gene assay for high-throughput endocrine disruptors screening.

关 键 词：p GL-4载体 报告基因实验 雌激素受体 LLC-MK2恒河猴肾细胞 荧光素酶 

分 类 号：R319[医药卫生—基础医学]