雄激素受体(hAR)CAT报告基因筛选AR激动剂和拮抗剂的方法研究

Study on the Method of Screening for AR Agonists and Antagonists by Human Androgen Receptor Reporter Gene Assay

[目的]建立雄激素受体(hAR)报告基因实验,为AR激动剂和拮抗剂活性筛选提供检测方法。[方法]构建受雄激素效应元件(ARE)调控的氯霉素乙酰转移酶(CAT)报告基因质粒pMMTV-CAT,将该质粒与hAR表达质粒AR/pcDNA3.1和阳性对照质粒pSV-β-Gal共转染非洲猴肾细胞株CV-1。以AR激动剂5α-双氢睾酮(5α-DHT)、糖皮质激素受体(GR)激动剂地塞米松和AR拮抗剂氟鲁他明为受试物,研究它们对AR的激动或拮抗作用。[结果]10^(-9)mol/L的5α-DHT显著诱导CAT的表达,诱导倍数为56.67,10^(-8)mol/L时达最大值,诱导倍数为79.30,在10^(-8)mol/L～10^(-6)mol/L范围内CAT的表达量维持在较高的平台水平。地塞米松不诱导CAT的表达。氟鲁他明在10^(-7)mol/L～10^(-5)mol/L浓度范围内显著拮抗1nmol/L的5α-DHT诱导的CAT的表达。[结论]5α-DHT对CAT的诱导和氰鲁他明对CAT的拮抗呈剂量-效应关系。该方法不存在与GR激动剂的交叉反应,具有较高的特异性。雄激素受体报告基因实验可以作为化学物(抗)雄激素活性筛选的方法。

[ Objective ] In order to provide a method of screening for androgen receptor agonists and antagonists, the human androgen receptor（ hAR ）reporter gone assay was developed. [ Methods ] The oligonucleotides containing androgen response element （ ARE ）were cloned into pCAT3-basic to create pMMTV-CAT. The CV-1 was cotranfected with three plasmids including the constructed pMMTV-CAT, the AR expression plasmid AR/pcDNA3.1 and the positive control plasmid pSV-β-Gal. AR agonism was performed by quantifying the induction of the reporter gone CAT product in response to activation of the AR by the referenc＇e androgen 5α-dihydrotestosterone （ 5α-DHT ）. AR antagonism was performed by measuring the ability of flutimide to inhibit the induction of the reporter gone CAT product by 5α-DHT. [ Results ] The CAT activity was induced obviously at 10^-9mol/L and the magnitude of the fold induction was 56.67 fold than that of the negative control. The maximal magnitude of the fold induction of 79.30 was achieved at 10^-8mol/L. The maximal effect contained at a plateau level at the ranges of 10^-8 mol/L-10^-6 mol/L. At the concentration ranges of 10^-7mol/L-10^-5 mol/L, flutamide significantly inhibited the CAT activity induced by 1 nmol/L 5α-DHT. The CAT activity could not be induced by the glucocorticoid receptor（ GR ）agonist dexamethasone at the tested dosages. [ Conclusion ] The assay displayed appropriate response to known AR agonist 5α-DHT and AR antagonist flutamide without cross-talk to GR agonist. The present study provided a tool of screening for AR agonists and antagonists.