建立基于荧光素报告基因系统检测雌激素样物质方法的研究  

Establishment of a cellular assay based on reporter gene for identification of strogenic compounds

作　　者：倪秉强[1] 段小群[2] 谭宁[2] 李军[2] 刘永明[3] 

机构地区：[1]广西医科大学第五临床学院,广西柳州545000 [2]桂林医学院科学实验中心,广西桂林541010 [3]桂林医学院生物技术学院,广西桂林541004

出　　处：《中国现代医学杂志》2009年第3期367-369,374,共4页China Journal of Modern Medicine

基　　金：桂林医学院医药生物技术重点学科建设基金

摘　　要：目的建立基于报告基因的HeLa细胞体外雌激素样物质检测方法。方法分别构建含有人雌激素hERα和hERβ编码序列的表达质粒以及人雌激素反应元件(estrogen response elements,ERE)调控的荧光素报告质粒,用脂质体将hERα、ERE质粒或hERβ、ERE质粒共转染HeLa细胞,用雌二醇(E2)处理后检测报告基因荧光素酶的活性。结果构建了pERαneo和pERβneo质粒以及ERE调控的荧光素酶报告质粒pGL3-ERE-Luc,共转染质粒pERE4-Luchygro和pERαneo或pERE4-Luchygro和pERαneo到HeLa细胞株后,建立稳定表达ERE-Luc的hERα/hERβHeLa细胞株,实验证实报告基因的表达与E2呈明显的剂量反应关系,ERα和ERβ的EC50分别为9.2×10-11mol/L和2.5×10-10mol/L。结论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。[Objective] To establish a cellular assay based on reporter gene for identification of estrogenic compounds. [Methods] Plasmids eneoding the two human estrogen receptor subtypes, pERαneo and pERβneo were construeted. Stably transfeeted estrogen-responsive HeLa cell lines were obtained by sequential transfeetion with pERF4-Luchygro and pERαneo or pERβneo plasmids. Then, Estradiol was added in cell culture media to check the activity of Luciferase. [Results] The pGL3-ERE-Lue plasmid was constructed, pGL3-ERE-Luc was transfeeted in HeLa cells stably expressed pERαneo or pERβneo In estradiol assay, there was a close relationship between lueiferase activities and E2 dose. The EC50 values for ERa and ERb were 9.2×10^（-11） mol/L and 2.5×10（-10） mol/L, respectively. [Conclusion] The cellular assay based on reporter gene for identification of estrogenic compounds is effective and reliable.

关 键 词：雌激素 报告基因 荧光素酶 

分 类 号：R347.913[医药卫生—基础医学]