【摘要】：目的建立和評價一種新的基于pGL-4載體的雌激素受體報告基因實驗系統(tǒng)。方法通過瞬時共轉染將雌激素受體表達載體、報告基因載體和內參載體轉入LLC-MK2細胞,用特定濃度受試物進行染毒并觀察其反應。結果 (1)細胞毒性試驗顯示,除雙酚A和壬基酚在100μmol/L時有細胞毒性外,其余受試物在設定濃度未見細胞毒性。(2)雌激素受體α報告基因實驗系統(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分別產生30.7倍和24.1倍對照組的熒光素酶表達,雙酚A、壬基酚和染料木黃酮均能誘導該系統(tǒng)熒光素酶的表達,他莫昔芬和染料木黃酮能抑制雌二醇誘導的熒光素酶表達。(3)雌激素受體β報告基因實驗系統(tǒng)中,雌二醇和己烯雌酚分別產生對照組14.4倍和5.6倍熒光素酶表達。結論基于pGL-4載體建立的雌激素受體報告基因實驗系統(tǒng)有著良好的重復性、靈敏度和特異度,且能檢測不同雌激素受體亞型的激動劑和拮抗劑,是一種新的用于內分泌干擾物大通量篩選的報告基因實驗系統(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校預防醫(yī)學教研室;西南醫(yī)科大學公共衛(wèi)生學院營養(yǎng)與食品衛(wèi)生教研室;四川大學華西公共衛(wèi)生學院(華西第四醫(yī)院);四川省宜賓衛(wèi)生學校預防教研室;
【基金】：國家自然科學基金重點項目(No.81030053)
【分類號】：Q78

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