基于CV-1细胞雌激素受体报告基因试验方法建立及双酚A类似物雌激素效应的检测

doi:10.11934/j.issn.1673-4831.2017.10.013

生态与农村环境学报 ›› 2017, Vol. 33 ›› Issue (10): 955-960.doi: 10.11934/j.issn.1673-4831.2017.10.013

• 研究方法 • 上一篇

基于CV-1细胞雌激素受体报告基因试验方法建立及双酚A类似物雌激素效应的检测

范德玲, 吉贵祥, 杨先海, 汪贞, 王蕾, 刘济宁, 石利利

环境保护部南京环境科学研究所, 江苏南京 210042

收稿日期:2016-11-03 出版日期:2017-10-25 发布日期:2017-11-24
通讯作者: 刘济宁,E-mail:ljn@nies.org E-mail:ljn@nies.org
作者简介:范德玲(1988-),女,安徽合肥人,助理研究员,硕士,从事环境内分泌干扰效应体外细胞研究。E-mail:fdl@nies.org
基金资助:
中央级公益性科研院所基本科研业务专项（20160407）；江苏省自然科学基金（BK20151100）

Establishment of CV-1 Cell-Based Estrogen Receptor Reporter Gene Assays Method and Determination of Estrogen Effect of Bisphenol A Analogues

FAN De-ling, JI Gui-xiang, YANG Xian-hai, WANG Zhen, WANG Lei, LIU Ji-ning, SHI Li-li

Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing 210042, China

Received:2016-11-03 Online:2017-10-25 Published:2017-11-24

摘要/Abstract

摘要：

以荧光素酶为报告基因，通过雌激素反应元件调控的带有荧光素酶报告基因质粒pERE-TATA-Luc和雌激素受体表达质粒rERa/pCI，采用瞬时共转染方法，将Luc报告基因质粒和受体表达质粒共转染至非洲猴肾CV-1细胞，以雌二醇作为阳性对照物，其浓度为10^-10 mol·L^-1时显著诱导荧光素酶的表达，浓度为10^-9 mol·L^-1时达最大值，半数效应浓度EC₅₀值为6.1×10^-11 mol·L^-1。通过雌二醇、己烯雌酚和木黄酮检测系统的灵敏性、有效性和稳定性验证，建立实验室稳定、灵敏的雌激素受体报告基因细胞系。基于非洲猴肾CV-1细胞受体报告基因试验，研究双酚A类似物拟雌激素干扰活性，雌激素活性大小顺序为BPAF > BPF > BPB > BPA > BPZ > BPAP > BPS > BPP。结果表明，CV-1细胞受体报告基因试验是一种筛选雌激素活性内分泌干扰物的快速、有效的方法。

关键词: 双酚A类似物, 雌激素效应, 体外试验

Abstract:

With luciferase being used as reporter gene, reporter gene plasmid with luciferase (pERE-TATA-Luc) was regulated by estrogen response element and estrogen receptor expression plasmid (rERa/pCI) was obtained. Using the transient co-transfection method, luciferase reporter gene plasmid and estrogen receptor expression plasmid were transfected into the kidney cells (CV-1) of an African monkey. Estradiol was used as CK for positive phenomenon. E₂ induced expression of luciferase significantly when it was 10^-10 mol·L^-1 in concentration and to the maximum when it was 10^-9 mol·L^-1 in concentration, and its EC₅₀ was 6.1×10^-11 mol·L^-1. Through validating sensitivity, efficacy and stability of the estradiol, diethylstilbestrol and genistein detecting systems, a stable sensitive estrogen receptor reporter gene cell line was established for laboratory. Based on the CV-1 receptor gene reporter assay, interferon activity of bisphenol A analogues type of estrogen was studied. In terms of estrogenic activity, a decreasing order of BPAF > BPF > BPB > BPA > BPZ > BPAP > BPS > BPP was found. All the findings demonstrate that the CV-1 cell receptor reporter gene assay is a rapid effective procedure for screening endocrine disrupting chemicals for estrogenic activity.

Key words: bisphenol A analogues, estrogenic activity, in vitro test

中图分类号:

X826

范德玲, 吉贵祥, 杨先海, 汪贞, 王蕾, 刘济宁, 石利利. 基于CV-1细胞雌激素受体报告基因试验方法建立及双酚A类似物雌激素效应的检测[J]. 生态与农村环境学报, 2017, 33(10): 955-960.

FAN De-ling, JI Gui-xiang, YANG Xian-hai, WANG Zhen, WANG Lei, LIU Ji-ning, SHI Li-li. Establishment of CV-1 Cell-Based Estrogen Receptor Reporter Gene Assays Method and Determination of Estrogen Effect of Bisphenol A Analogues[J]. Journal of Ecology and Rural Environment, 2017, 33(10): 955-960.

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基于CV-1细胞雌激素受体报告基因试验方法建立及双酚A类似物雌激素效应的检测

Establishment of CV-1 Cell-Based Estrogen Receptor Reporter Gene Assays Method and Determination of Estrogen Effect of Bisphenol A Analogues

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