基于荧光素酶报告基因的MCF7细胞雌激素样物质检测方法的建立

Establishment of a reporter gene-based assay for identification of estrogenic compounds in MCF7 cells

目的:建立基于报告基因的MCF7细胞体外雌激素样物质检测方法,并研究2,2-双(对氯苯基)-1,1,1-三氯乙烷(2,4-DDT)和甲氧氯(METH)的雌激素样作用。方法:合成人雌激素反应元件(ERE)核心片段并将其插入pGL3-promoter载体的多克隆位点,构建成雌激素反应元件ERE调控的报告质粒pERE-Luc,用脂质体转染法将pERE-Luc及内对照质粒phRL-SV40瞬时转染MCF7细胞,用17β-雌二醇(E2)及受体拮抗剂三苯氧胺(TAM)等处理后检测报告基因荧光素酶的表达,并用雌激素样物质2,4-DDT和METH对方法的有效性进行验证。结果:构建了ERE调控的报告质粒pERE-Luc,转染pERE-Luc的MCF7细胞报告基因的表达与E2呈明显的剂量反应关系,1×10-11mol/L E2可引起报告基因表达,至1×10-9mol/L可达到最大表达值,而未转染pERE-Luc时与E2无明显反应,TAM可以显著抑制E2引起的报告基因的表达。验证结果表明,2,4-DDT浓度>1×10-7mol/L及METH浓度>1×10-6mol/L时可产生雌激素样活性,2,4-DDT的雌激素样活性强于METH。结论:建立的基于荧光素酶报告基因的体外雌激素样物质检测方法有效可靠,以此方法检测2,4-DDT和METH显示有明显的雌激素样活性作用。

Objective：To establish a reporter gene-based assay for identification of estrogenic compounds in MCF7 cells and study the estrogenic effects of 2,4-DDT and methoxychlor（METH）. Methods： pERE-Luc plamid was generated by inserting estrogen response element （ERE） fragment into MCS of pGL3-promoter vector. MCF7 cells were cotransfected with pERE-Luc and phRL-SV40 using Sofast transfection reagent. The cells were then treated with 17β-estradiol （E2）, tamoxifen （Tam）, 2,4-DDT and METH and expression of the reporter gene in the cell lysates was assayed using Dual-Luciferase reporter assay system. Results： The pERE-Luc plasmid was constructed. Luciferase activities of pERE-Luc-transfected MCF7 cells showed a dose-dependent relationship with E2. E2 at 1×10^-11 mol/L induced the expression of reporter gene and at 1×10^-9mol/L resulted in the peak luciferase activity. E2 did not induce luciferase activity without pERE-Luc and Tam inhibited E2-induced luciferase expression. 2,4-DDT induced the luciferase activity at 〉 1×10^-7 mol/L and METH at 〉 1×10^-6 mol/L. The estrogenie activity of 2,4-DDT was more potent than that of METH. Conclusion： The reporter gene-based assay we established is effective and reliable; 2,4-DDT and METH demonstrate obvious estrogenic activities when detected by our method.