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

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。

With luciferase being used as reporter gene, reporter gene plasmid with luciferase （ pERE - T A T A - L u c） was regulated by estrogen response element and estrogen receptor expression plasmid （rERa/p C I） 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 m o n k e y. Estradiol was used as C K for positive phenomenon. E2 in-duced expression of luciferase significantly w h e n it was 10 10 mol ＊ L 1 in concentration and to the m a x i m u m w h e n it was 10 9 mol ＊L 1 in concentration, and its E C 50 was 6.1x 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 C V -1 receptor gene reporter assay, interferon activity of bisphenol A ana-logues 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 C V -1 cell receptor reporter gene assay is a rapid effective procedure for screening endocrine disrupting chemicals for estrogenic activity.