报告基因方法对壬基酚、双酚A雌激素样活性的体外研究

Detection of estrogenic effects of nonylphenol and bisphenol A in vitro reporter gene-based assays

目的利用基于报告基因的MCF7细胞体外雌激素检测方法对壬基酚(NP)、双酚A(BPA)的雌激素样活性及作用机制进行研究。方法合成人雌激素反应元件(ERE)核心片断并将其插入pGL3promoter载体的多克隆位点构建而成雌激素反应元件ERE调控的报告质粒pERELuc,用脂质体转染法将pERELuc及内对照质粒phRLSV40瞬时转染MCF7细胞,用17β雌二醇(E2)、三苯氧胺(Tam)、壬基酚、双酚A等处理后检测报告基因荧光素酶的表达。结果转染pERELuc的MCF7细胞的报告基因表达与E2呈明显的剂量-反应关系,1×10-11molLE2可引起报告基因的表达,至1×10-9molL可达到最大表达值,而未转染pERELuc时与E2无明显反应,三苯氧胺可以显著抑制E2引起的报告基因的表达。1×10-6molL以上NP和BPA出现雌激素样作用。NP的雌激素样活性略强于BPA。结论本试验建立并采用的基于报告基因的体外雌激素检测方法是可行的,NP和BPA有一定的雌激素样活性作用。

Objective To explore the estrogenic effects and disruptive mechanism of NP and BPA by reporter genebased assays we developed. Methods pERE-Luc plamid was generated by inserting estrogen response element （ERE） fragment into MCS of pGI3-promoter vector. MCF7 cells were cotransfected with pERE-Luc and phRL-SV40 using Sofast transfection reagent. The cells then treated with 17β-estradiol （E2）, tamoxifen （Tam）, nonylphenol（NP） and bisphenol A （BPA） and expression of the repoter gene in the cell lysates was assayed using Dual-Lucferase reporter assay system. Results The pERE-Luc plasmid was constructed, l,uciferase activities of MCF7 cells transfected pERE-Luc showed doseresponed realitionship with E2. 1×10^-11mol/L E2 could induce the expression of reporter gene and 1×10^-9 mol/L E2 resulted in the largest luciferase activity. E2 couldn＇t induce the luciferase activity without pERE-Luc. Tam is a complete antagonist, inhibited the E2-induced luciferase expression. NP induced the luciferase activity at concertrations 〉 1×10^-6mol/L, BPA induced the luciferase activity at concertrations 〉 1×10^-6mol/L. The estrogenic activity of NP was more than BPA. Conclusion The assay we established is usful, NP and BPA showed estrogenic activities.