甲状腺激素受体介导的荧光素酶报告基因试验筛查内分泌干扰化学物

The Thyroid Hormone Receptor Mediated Luciferase Reporter Gene Assays Screening for EDCs

目的采用人源性甲状腺激素受体(TRα/β)的荧光素酶报告基因试验系统筛查内分泌干扰化学物(EDCs),评估双酚A(BPA)、甲萘威和1-萘酚(1-NAP)的拟/抗甲状腺激素活性。方法以恒河猴肾细胞(LLCMK2)作为转染细胞,通过瞬时转染的方法分别构建基于pGL3-promega和pGL4.27的TRα/β的报告基因试验。用三碘甲状腺氨酸(T3)、甲状腺氨酸(T4)作为阳性受试物评价两个检测系统的灵敏性,并检测BPA、甲萘威和1-NAP的拟/抗甲状腺激素活性。结果基于pGL3-promega的TRβ的报告基因试验,T3的最低检测限为1.216×10^(-11) mol/L,在7.482×10^(-6) mol/L时诱导荧光素酶(Luc)的表达倍数是对照组的5.98倍,半数有效浓度(EC_(50))为3.327×10^(-8) mol/L;T4最低检测限为1.622×10^(-8) mol/L,最大诱导Luc表达的倍数为3.4倍,EC_(50)为2.213×10^(-7) mol/L。基于pGL4.27的TRβ的报告基因试验中,T3的最低检测限为9.863×10-12 mol/L,在1.671×10^(-6) mol/L时产生最大诱导Luc表达的倍数为对照组的8.57倍,EC_(50)为3.327×10^(-8) mol/L;T4最低检测限为1.349×10^(-9) mol/L,最大诱导Luc表达的倍数是4.6倍,EC_(50)为4.074×10^(-7) mol/L。用TRβ的报告基因试验系统评价BPA、甲萘威和1-NAP都无甲状腺受体激动剂活性,甲萘威和1-NAP有一定受体拮抗性。结论本研究建立的基于pGL3-promega和pGL4.27的TRβ的报告基因试验都有较高的灵敏性,pGL4.27相对更高,可以用来筛查内分泌干扰化学物,检测化学物质的拟/抗甲状腺激素活性。

Objective This study in order to use report gene assay based on the thyroid hormone receptor （TR） α/β from human origin for screening endocrine disruptors chemicals （EDCs）, evaluating the thyroid hormone activity of Bisphenol （BPA）, 1-Naphthaleny methyl carbamate and 1-naphthol （1-NAP）. Methods Using Rhesus monkey kidney cells （LLC-MK2） as transfection cell to establish the gene report assay system based on pGL-3-promega and pGL4.27 of TRα/β through the method of transient transfection. Using T3 and T4 as positive subjects to evaluation the effectiveness of two detection systems and detect the thyroid hormone activity of BPA, 1-Naphthaleny methyl carbamate, 1-NAP. Results The TRβ LLC-MK2 report gene assay based on pGL3-promega, the minimum detectable limit of T3 is 1.216×10^-11 mol/L, the largest induction multiple was shown at 7.482×10^-6 mol/L, the expression multiple of induced Lucifrerasewas 5.98-fold that of the vehicle control, the EC50 was 3.327×10^-8 mol/L; The minimum detectable limit of T4 was 1.622×10^-8 mol/L, the largest induction Luc expression was 3.4-fold of vehicle control, the EC50 was 2.213×10^-7 mol/L. The TRβ LLC-MK2 report gene assay based on pGL4.27, the minimum detectable limit of T3 was 9.863×10^-12 mol/L, the largest induction Luc expression as shown at 1.671×10^-6 mol/L, resulting in 8.57-fold of vehicle control, the EC50 is 3.327×10^-8 mol/L. The minimum detectable limit of T4 was 1.349×10^-9mol/L, the largest induction Luc expression was 4.6-fold of vehicle control, the EC50 is 4.074×10^-7 mol/L. There was no thyroid hormone activity by using TRβ report gene assay to evaluate BPA, 1-Naphthaleny methyl carbamate or 1-NAP, but 1-Naphthaleny methyl carbamate and1-NAP have some degree receptor antagonism. Conclusion The TRβ LLC-MK2 report gene assay based on pGL3-] promega and pGL4.27 show highly sensitive （pGL4.27 relatively higher）, can be used to screen for EDCS and test chemical thyroid hormone activity effectively.