Zhao Hai-Tao, Zhang Tian-Bao, Zhu Yong-Fei, Wan Xu-Ying, Zhu Yuping, Ma Xili
Department of Toxicology, Second Military Medical University, Shanghai 200433, China.
Wei Sheng Yan Jiu. 2006 Jan;35(1):13-5.
To explore the estrogenic effects and disruptive mechanism of NP and BPA by reporter gene-based assays we developed.
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 then treated with 17beta-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.
The pERE-Luc plasmid was constructed. Luciferase activities of MCF7 cells transfected pERE-Luc showed dose-responed realitionship with E2. 1 x 10(-11) mol/L E2 could induce the expression of reporter gene and 1 x 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 x 10(-6) mol/L, BPA induced the luciferase activity at concertrations > 1 x 10(-6) mol/L. The estrogenic activity of NP was more than BPA.
The assay we established is usful, NP and BPA showed estrogenic activities.
通过我们开发的基于报告基因的检测方法，探讨壬基酚（NP）和双酚A（BPA）的雌激素效应及干扰机制。
通过将雌激素反应元件（ERE）片段插入pGL3-启动子载体的多克隆位点（MCS）构建pERE-Luc质粒。使用Sofast转染试剂将pERE-Luc和phRL-SV40共转染MCF-7细胞。然后用17β-雌二醇（E2）、他莫昔芬（Tam）、壬基酚（NP）和双酚A（BPA）处理细胞，使用双荧光素酶报告基因检测系统检测细胞裂解物中报告基因的表达。
构建了pERE-Luc质粒。转染pERE-Luc的MCF-7细胞的荧光素酶活性与E2呈剂量反应关系。1×10⁻¹¹ mol/L E2可诱导报告基因表达，1×10⁻⁹ mol/L E2导致最大荧光素酶活性。无pERE-Luc时E2不能诱导荧光素酶活性。Tam是完全拮抗剂，抑制E2诱导的荧光素酶表达。NP在浓度>1×10⁻⁶ mol/L时诱导荧光素酶活性，BPA在浓度>1×10⁻⁶ mol/L时诱导荧光素酶活性。NP的雌激素活性大于BPA。
我们建立的检测方法有用，NP和BPA表现出雌激素活性。
