基于ERC-PCR的环境内分泌干扰物检测体系研究

ERC-PCR based detection for environmental endocrine-disrupting compounds

目的利用雌激素与雌激素受体α(estrogen receptorα,ERα)间基于配体受体激活效应的生物学原理和探针的定量PCR扩增-实时荧光检测技术,建立痕量环境内分泌干扰物(endocrine disrupting compounds,EDCs)的快速检测体系。方法以2条含雌激素反应元件(estrogen response elements,ERE)并与ERα有高亲和力的DNA序列COX7RP-ERE和VitA2-ERE,作为结合探针,进行ERα体外受体-配体特异结合,形成配体-ERα-DNA探针复合体(ERα-DNA probe com-plex,ERC),荧光定量检测ERE含量,并考察17β-雌二醇(E2)、雌酮(E1)、己烯雌酚(DES)及邻苯二甲酸二甲酯(DMP)的结合情况。结果与VitA2-ERE相比,人源性COX7RP-ERE在ERα体外受体-配体结合过程中具有更好的结合效应;E1、E2、DES、DMP 4种雌激素类化合物对COX7RP-ERE的结合存在明显的剂量-效应关系,其对COX7RP-ERE结合效应的半数有效浓度(EC50)分别为0.89 nmol/L、19.9 pmol/L、0.79 nmol/L、6.31 pmol/L。结论检测体系中结合探针的结合量存在明显的雌激素剂量依赖性,这种基于ERα激活效应的ERC-PCR定量检测体系可以用于监测化合物和EDCs的类雌激素活性。

Objective To develop a rapid and high-throughput screening system to detect trace environmental endocrine disrupting compounds （EDCs） in vitro based on the estrogen-dependent binding of estrogen receptors α （ERos） with estrogen response element （ERE）. Methods Two DNA sequences, COX7RP and VitA2 gene promoter which contained ERE and were of high affinity to the ERα, were used as combination probes to measure the estrogenic concentration in the estrogen-dependent ERα-ERE binding process. The combination probe was detected by real-time fluorescent quantitative PCR. Five compounds, 17β-estradiol （E2 ） , estrone （E1）, diethylstilbestrol （DES） and dimethylphthalate （DMP） at the final concentration of 10^-14 to 10^-6 mol/L were detected by the present method. Results Compared with the VitA2-ERE, human-derived COX7RP-ERE had a better combination with the probe during estrogen-dependent ERα-ERE binding process in vitro. Our present detection system provided sensitive estrogen-dependent dose-response curves for E1, E2 , DES and DMP, with median effective concentration （EC50） at 0.89 nmol/L, 19.9 pmol/L, 0.79 nmol/L, and 6.31 pmol/L, respectively. Conclusion Our detection system based on ERC-PCR strategy is a sensitive, simple, and rapid tool for the detection of EDCs.