基于荧光素酶报告基因HeLa细胞雌激素检测方法的建立及其在水产品检测中的应用

Establishment of a Reporter Gene-based Assay for Identification of Estrogenic Compounds in HeLa Cells and Its Application in Detection of Aquatis Products

利用荧光素酶(Luciferase)报告基因检测技术,构建稳定表达人雌激素受体α(hERα)或人雌激素受体β(hERβ)的重组HeLa细胞株(该细胞株无内源性激素受体)。通过培养已转入并能成功表达的包含雌激素反应元件ERE的报告质粒pERE-Luc的HeLa细胞,用17β-雌二醇(E2)、己烯雌酚、孕酮处理后检测荧光素酶的表达活性。Luc的荧光表达倍率与E2、己烯雌酚、孕酮标样浓度呈非线性关系,相关系数分别达到0.879 8、0.955 0和0.926 2。1.0×10-13mol/L时E2可出现明显的兴奋效应,至1.0×10-9mol/L可达到最大反应值,而未转染雌激素受体质粒与E2无明显反应。同时己烯雌酚和孕酮均可以表现雌激素样活性,浓度大于1.0×10-10mol/L,己烯雌酚表现雌激素样活性;浓度大于1.0×10-11mol/L,孕酮表现雌激素样活性,表明孕酮的雌激素样活性高于己烯雌酚。使用浓度均为1.0×10-10mol/L的E2、己烯雌酚、孕酮分别处理,报告基因的发光均值分别为0.947 4、0.312 3和0.579 6,相对标准偏差(RSD)分别为13.7%、3.7%和3.4%,证明上述3种物质对于荧光素酶报告基因表达活性的影响具有很好的重复性。运用同样原理检测草鱼、带鱼、鲫鱼和鲷鱼鱼肉中雌激素样物质的含量,E2的加标回收率均在75%以上,RSD为7.3%～11.6%,精密度较好。该研究基于荧光素酶报告基因的体外雌激素样物质检测方法有效可靠。

Based on a recombinant cell strain for high sensitivity detection of estrogenic activity, estrogen synthesis technology is has the advantages without endogenous or estrogen receptor a revolutionary technology of the detection of the biological efficacy. It of high efficiency and high sensitivity characteristic. The HeLa cells （the cells hormone receptor） were used to recombine the human estrogen receptor a（hERa） β（hERβ）, and then build a stable expression of cell to detect the luciferase report gene. pERE -Luc plamid was generated by inserting estrogen response element （ERE） fragment into MCS of pGL3 - promoter vector. HeLa cells were cotransfected with pERE - Luc using so fast transfection reagent. The cells were then treated with 17β-estradiol（E2）, diethylstilbestrol and pro- gesterone, and expression of the reporter gene in the cell lysates was assayed using Dual - Luciferase reporter assay system. The pERE - Luc plasmid was constructed. Luciferase activities of pERE - Luc-ransfected HeLa cells showed that the correlations between RFU of Luc activity and E2, diethylstilbestrol and progesterone standard sample concentration was nonlinear, r2 was 0. 879 8, 0. 955 0 and 0. 926 2, respectively. E2 at 1.0 × 10^-13 mol/L induced the expression of reporter gene and that at 1.0 ×10^ -9 mol/L resulted in the peak luciferase activity. Diethylstilbestrol luciferase activity was larger than 1.0 ×10^-10 mol/L and progesterone was larger than 1.0 ×10^-11 mol/L. The estrogenic activity of progesterone was more potent than that of diethylstilbestrol. The averages of the reportergene emitted were 0. 947 4, 0. 312 3 and 0. 579 6, the relative standard deviation were 13.7% , 3.7% and 3.4% after treated with E2, diethylstilbestrol and progesterone at 1.0 × 10 ^-10 mol/L. Results showed that good quality reprodueibilities of lueiferase reporter gene expression were obtained after treated with E2, diethylstilbestrol and progesterone. The spiked recoveries of E2 were over 75% in four fishes and the relative standard deviations （RSDs） were between 7.3% and 11.6% The proposed reporter gene-based assay is effective and reliable.