环境化学物雄激素干扰活性检测方法的建立

Establishment of detecting assay for evaluating the androgenic disrupting activity of environmental chemicals

目的:建立雄激素受体(AR)介导的荧光素酶(Luc)报告基因试验方法,为化学物拟/抗雄激素活性的检测提供研究工具。方法:构建受雄激素反应元件调控的荧光素酶报告基因质粒pMMTV-Luc,将该质粒与表达AR的质粒AR/pcDNA3.1和阳性对照质粒phRL-tk共转染CV-1细胞,建立AR报告基因试验。以5α-双氢睾酮(5α-DHT)为阳性对照检测该方法的筛选效率。CV-1转染后用受试化学物染毒,根据Luc表达的变化,判断化学物的雄激素干扰活性。结果:5α-DHT对共转染三种质粒的CV-1的诱导作用表现为:10-10M的5α-DHT显著诱导Luc的表达,10-7M时达最大值,诱导倍数为61.83倍,计算其半数效应浓度EC50为3.65 nM。糖皮质激素受体(GR)激动剂地塞米松(dexamethasone)不诱导Luc的表达。AR拮抗剂氟他胺(flutamide)在10-7M～10-5M浓度范围内显著拮抗1nM的5α-DHT诱导的Luc的表达,其半数抑制浓度(IC50)为3.49μM。BPA在10-6M和10-5M时能拮抗1 nM 5α-DHT诱导的Luc的表达,其IC50为2.14μM。结论:本研究建立的AR报告基因试验具有较高的灵敏度和特异性,化学物BPA具有抗雄激素活性。

Objective：In order to provide a model for screening the（anti）androgen effects of chemicals,an AR reporter gene assay based transient transfection was developed using the Luc as the reporter gene.Methods：Reporter plasmid pMMTV-Luc containing the Luc regulated by ARE were constructed,and then transfected with expression plasmid AR/pcDNA3.1 containing the cDNA of AR,as well as the control plasmid phRL-tk to create the AR reporter gene assay.The reference androgen 5α-dihydrotestosterone（5α-DHT） was used to verify the performance of the assay.Androgenic disrupting activity of chemicals was generally performed by quantifying the induction of the reporter gene Luc product in response to activation or inhibition of the AR by the test chemicals.Results：The AR mediated reporter gene assay could detecte the effects cause by 10-10M 5α-DHT;at the concentration of 10-7M,the maximal magnitude of the fold induction of 61.83-fold of control was achieved and maintained at a plateau level at the ranges of 10-7M-10-6M.The median effective concentration（EC50） value of 5α-DHT was 3.65 nM.GR agonist dexamethosone could not induce the expression of Luc.At the concentration ranges of 10-7M-10-5M,flutamide significantly inhibited the Luc activity induced by 1nM 5α-DHT.The median inhibitory concentration（IC50） value was 3.49 μM.BPA could suppress the function of 1nM 5α-DHT,with IC50 of 2.14 μM.Conclusion：The assay showed high sensitivity and acceptable repeatability to 5α-DHT.BPA possessed the anti-androgenic activity.