雌激素受体亚型介导的荧光素酶报告基因试验方法的建立及应用

目的 建立人源性雌激素受体（hERα/β）介导的荧光素酶报告基因试验方法，比较不同雌激素受体亚型介导的报告基因试验的反应性和灵敏度，为检测内分泌干扰物拟雌激素活性提供研究工具。方法 以恒河猴肾细胞（LLC-MK2）为转染细胞，以萤火虫荧光素酶基因（Luc）为报告基因，利用瞬时转染的方法，将表达hERα/hERβ的质粒与重组Luc报告基因质粒pERE-minP-Luc2P、内对照质粒pGL4.74共转染LLC-MK2细胞，建立由hERα/β介导的Luc报告基因试验方法。以雌二醇(E 2)、己烯雌酚(DES)作为阳性受试物，以10-5 mol/L 雌激素拮抗剂（ICI 182,780）和不同浓度的E 2共同染毒，观察Luc表达的变化。用双酚A（BPA）和染料木黄酮（GS）对方法的有效性进行检验并比较雌激素受体两亚型反应性、灵敏度和特异性。结果 hERα报告基因系统对E 2的最低检测限为1.9×10-11 mol/L，在10-8 mol/L处获得最高诱导倍数，是对照组的30.7倍。hERβ报告基因系统对E 2的最低检测限为2.2×10-11 mol/L，在10-8 mol/L处获得最高诱导倍数，是对照组的14.4倍。ICI 182,780在10-5 mol/L浓度下能显著抑制两个报告基因系统E 2的雌激素活性。未转染受体表达载体hER-pcDNA3.1的LLC-MK2细胞用不同浓度E 2诱导，两个报告基因系统均与E 2无明显反应。BPA和GS在以上两个不同的报告基因系统中均能诱导Luc表达，但同一受试物在不同雌激素亚型介导的报告基因系统中表达倍数不同。结论 本研究建立ER不同亚型的报告基因试验有较高的灵敏度和重复性。hERα介导的报告基因试验灵敏度高于hERβ，在hERα系统中BPA雌激素活性强于GS，在hERβ系统中GS雌激素活性强于BPA。

Estrogen Receptor Subtype-mediated Luciferase Reporter Gene Assays for Determining (anti) Estrogen Effect ofChemicals

Objective To establish gene assays for determining (anti) estrogen effect of environmental chemicals; and to compare the reactivity and sensitivity of two assays with different estrogen subtype. Methods Human estrogen receptor α (hERα) and hERβ mediated reporter gene assays employing firefly luciferase (Luc) were developed. The expression plasmid hERα or hERβ was constructed and transiently co-transfected into LLC-MK2 cells with pERE-minP-Luc2P reporter plasmid and the control plasmid pGL4.74. Estradiol (E 2) and diethylstilbestrol (DES) served as positive test substances to verify the performance of the assays. The effectiveness of the assays for detecting anti-estrogenic activity was tested using 10-5 mol/L ICI 182,780 under different concentrations of E 2. The performance of the two subtype-mediated assays was verified and compared using bisphenol A (BPA) and genistein (GS). Results The hERα mediated assay found expression of reported gene at 1.9×10-11mol/L E 2; and the largest luciferase activity was shown at 10-8 mol/L E 2, resulting in 30.7-fold of vehicle control. The hERβ mediated assay found expression of reporter gene at 2.2×10-11 mol/L E 2, and the largest luciferase activity was shown at 10-8 mol/L E 2, resulting in 14.4-fold of vehicle control. ICI 182,780 inhibited estrogenic activity of E 2 significantly. In both assays, E 2 failed to induce luciferase activity without hER-pcDNA3.1. BPA and GS induced luciferase activity. Conclusion Both assays have high sensitivity and reproducibility for detecting (anti) estrogen effect. The pGL4-based hERβ has lower sensitivity than the hERα-mediated reporter gene assay. BPA shows stronger estrogenic activity than GS in hERα mediated reporter gene assay; whereas, GS shows stronger estrogenic activity than BPA in hERβ mediated reporter gene assay.