【摘要】：目的建立和評價(jià)一種新的基于pGL-4載體的雌激素受體報(bào)告基因?qū)嶒?yàn)系統(tǒng)。方法通過瞬時(shí)共轉(zhuǎn)染將雌激素受體表達(dá)載體、報(bào)告基因載體和內(nèi)參載體轉(zhuǎn)入LLC-MK2細(xì)胞,用特定濃度受試物進(jìn)行染毒并觀察其反應(yīng)。結(jié)果 (1)細(xì)胞毒性試驗(yàn)顯示,除雙酚A和壬基酚在100μmol/L時(shí)有細(xì)胞毒性外,其余受試物在設(shè)定濃度未見細(xì)胞毒性。(2)雌激素受體α報(bào)告基因?qū)嶒?yàn)系統(tǒng)最低檢測限為1.9×10~(-11)mol/L,雌二醇和己烯雌酚在10~(-8)mol/L分別產(chǎn)生30.7倍和24.1倍對照組的熒光素酶表達(dá),雙酚A、壬基酚和染料木黃酮均能誘導(dǎo)該系統(tǒng)熒光素酶的表達(dá),他莫昔芬和染料木黃酮能抑制雌二醇誘導(dǎo)的熒光素酶表達(dá)。(3)雌激素受體β報(bào)告基因?qū)嶒?yàn)系統(tǒng)中,雌二醇和己烯雌酚分別產(chǎn)生對照組14.4倍和5.6倍熒光素酶表達(dá)。結(jié)論基于pGL-4載體建立的雌激素受體報(bào)告基因?qū)嶒?yàn)系統(tǒng)有著良好的重復(fù)性、靈敏度和特異度,且能檢測不同雌激素受體亞型的激動(dòng)劑和拮抗劑,是一種新的用于內(nèi)分泌干擾物大通量篩選的報(bào)告基因?qū)嶒?yàn)系統(tǒng)。
[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 偽 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.
【作者單位】： 重慶醫(yī)藥高等�？茖W(xué)校預(yù)防醫(yī)學(xué)教研室;西南醫(yī)科大學(xué)公共衛(wèi)生學(xué)院營養(yǎng)與食品衛(wèi)生教研室;四川大學(xué)華西公共衛(wèi)生學(xué)院(華西第四醫(yī)院);四川省宜賓衛(wèi)生學(xué)校預(yù)防教研室;
【基金】：國家自然科學(xué)基金重點(diǎn)項(xiàng)目(No.81030053)
【分類號】：Q78

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