改良的环境化学物雌激素干扰活性检测方法的建立和应用  

Establishment of an improved detecting assay for evaluating the estrogenic disrupting activity of environmental chemicals

作　　者：司朝宗[1] 孙宏[2] 甄士祺[2] 杨海兵[1] 陈晓东[2] 王彩生[2] 

机构地区：[1]江苏省苏州市疾病预防控制中心,苏州215004 [2]江苏省疾病预防控制中心,南京210009

出　　处：《江苏预防医学》2012年第4期8-10,共3页Jiangsu Journal of Preventive Medicine

基　　金：国家自然科学基金青年基金(30901222)

摘　　要：目的建立雌激素受体(ER)介导的荧光素酶(Luc)报告基因试验方法,为化学物拟(抗)雌激素活性的检测提供研究工具。方法将受雌激素反应元件调控的荧光素酶报告基因质粒pERE-aug-Luc和表达ER的质粒rERα/pCI转染Vero细胞,建立ER报告基因试验。以雌激素(E2)为阳性对照检测该方法的筛选效率。Vero转染后用受试化学物染毒,根据Luc表达的变化,判断化学物的雌激素干扰活性。结果雌激素对转染两种质粒的Vero的诱导作用表现为:10-7 mg/L的雌激素显著诱导Luc的表达,最大诱导倍数为100.959倍,半数效应浓度EC50为3.57×10-6 mg/L。化学物BPA可诱导Luc的表达,其中在0.01mg/L时即可显著性的诱导Luc表达,未发现BPA具有雌激素拮抗作用。结论本研究建立的ER报告基因试验具有较高的灵敏度和特异性,化学物BPA具有拟激素活性。Objective In order to provide a model for screening the （anti）estrogen effects of chemicals,an ER reporter gene assay based transient transfection was developed using the Luc as the reporter gene. Methods Reporter plasmid pERE-aug- Luc containing the Luc regulated by ERE and expression plasmid rER /pCI containing the eDNA of ER were transfected into Vero to create the ER reporter gene assay. The reference E2 was used to veryfy the performance of the assay. Estrogenic disrupting activity of chemicals was generally performed by quantifying the induction of the reporter gene Luc product in response to activation or inhibition of the ER by the test chemicals. Results The ER mediated reporter gene assay could detecte the effects caused by 10-7 mg/L E2; the maximal fold induction was 100. 959-fold of control and maintained at a plateau level at the ranges of 10-5 mg/L -10 3 mg/L. The median effective concentration （EC50） value of estrogen was 5.37 ×10-6 mg/L. BPA could induce the expression of Lue. At the concentration of 0.01 mg/L,BPA could induce the expression of Luc significantly,no antiestrogenic effect of BPA were found. Conclusion The assay showed high sensitivity and acceptable repeatability to ER. BPA possessed the estrogenic activity.

关 键 词：内分泌干扰物 雌激素干扰效应 转录激活试验 

分 类 号：R33[医药卫生—人体生理学]