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
发布时间：2018-08-26 19:35
【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。
[Abstract]:Objective to establish and evaluate a novel estrogen receptor reporter gene system based on pGL-4 vector. Methods the expression vector of estrogen receptor was transfered into LLC-MK2 cells by transient cotransfection, and the reporter gene vector and internal reference vector were transfected into LLC-MK2 cells. Results (1) Cytotoxicity test showed that bisphenol A and nonylphenol had cytotoxicity at 100 渭 mol/L. (2) the lowest detection limit of estrogen receptor 伪 reporter gene system was 1.9 脳 10 ~ (-11) mol/L, estradiol and diethylstilbestrol in 10 ~ (-8) mol/L. Bisphenol A, nonylphenol and genistein could all induce the expression of luciferase. Tamoxifen and genistein could inhibit the expression of luciferase induced by estradiol. Estradiol and diethylstilbestrol produced 14.4 fold and 5.6 fold luciferase expression respectively. Conclusion the estrogen receptor reporter gene system based on pGL-4 vector has good reproducibility, sensitivity and specificity, and can detect agonists and antagonists of different estrogen receptor subtypes. It is a new report gene experiment system for endocrine disruptor screening.
【作者单位】： 重庆医药高等专科学校预防医学教研室;西南医科大学公共卫生学院营养与食品卫生教研室;四川大学华西公共卫生学院(华西第四医院);四川省宜宾卫生学校预防教研室;
【基金】：国家自然科学基金重点项目(No.81030053)
【分类号】：Q78
本文编号：2205947
[Abstract]:Objective to establish and evaluate a novel estrogen receptor reporter gene system based on pGL-4 vector. Methods the expression vector of estrogen receptor was transfered into LLC-MK2 cells by transient cotransfection, and the reporter gene vector and internal reference vector were transfected into LLC-MK2 cells. Results (1) Cytotoxicity test showed that bisphenol A and nonylphenol had cytotoxicity at 100 渭 mol/L. (2) the lowest detection limit of estrogen receptor 伪 reporter gene system was 1.9 脳 10 ~ (-11) mol/L, estradiol and diethylstilbestrol in 10 ~ (-8) mol/L. Bisphenol A, nonylphenol and genistein could all induce the expression of luciferase. Tamoxifen and genistein could inhibit the expression of luciferase induced by estradiol. Estradiol and diethylstilbestrol produced 14.4 fold and 5.6 fold luciferase expression respectively. Conclusion the estrogen receptor reporter gene system based on pGL-4 vector has good reproducibility, sensitivity and specificity, and can detect agonists and antagonists of different estrogen receptor subtypes. It is a new report gene experiment system for endocrine disruptor screening.
【作者单位】： 重庆医药高等专科学校预防医学教研室;西南医科大学公共卫生学院营养与食品卫生教研室;四川大学华西公共卫生学院(华西第四医院);四川省宜宾卫生学校预防教研室;
【基金】：国家自然科学基金重点项目(No.81030053)
【分类号】：Q78
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