卵巢颗粒细胞中胰岛素和雄激素对芳香化酶和5α-还原酶1影响的研究

目的研究卵巢颗粒细胞中胰岛素对睾酮激素转化过程的影响。方法选用人卵巢颗粒细胞瘤样细胞系(KGN)作为研究对象。应用RT-PCR分析KGN细胞在梯度睾酮(1.25、2.5、5、10、20ng/ml)及胰岛素(1、10、100ng/ml)处理4h后CYP19a1和5RD5A1mRNA水平;Western-blot检测高浓度睾酮(10、20ng/ml)及梯度胰岛素处理KGN细胞24h后芳香化酶和5α-还原酶1蛋白水平;同步收集上清,应用放射性免疫法及酶联免疫法检测培养液上清睾酮、雌二醇及双氢睾酮的浓度。结果胰岛素剂量依赖性地上调CYP19a1的表达和活性,增加睾酮向雌二醇的转化;同时抑制5RD5A1的表达和活性,抑制睾酮向双氢睾酮的转化;转化比例改变但消耗水平不变,导致睾酮的异常累积。结论高雄激素环境下,胰岛素可通过影响睾酮转化酶促使睾酮转化异常。本实验为高胰岛素及高雄激素水平在卵巢微环境相互作用加剧PCOS病程提出了可能的机制。

Double-faced functions of insulin on CYP19a1 and 5RD5A1 under hyperandrogenism in ovarian granulosa cell

Objective:To explore the effect of insulin on the aromatase (CYP19A1) and 5α-reductase1 (5RD5A1) under hyperandrogenism during the process of testosterone (T) transformation.Methods:The mRNA expression levels of CYP19A1 and 5RD5A1 were measured by quantitative real-time PCR after 4 hours treatment of gradient insulin(1,10,100 ng/ml)and T (1.25,2.5,5,10,20 ng/ml) in KGN cells,and the protein expression of aromatase and 5α-reductase1 were detected by Western blot.Meantime,the supernatants of the cells were collected,and then T and estradiol(E 2) levels were detected by radioactive immunoassay (RIA),and dihydrotestosterone (DHT) was measured with enzyme linked immunosorbent assay (ELISA).Results:Insulin enhanced the aromatase expression in a concentration dependent manner.And this effect was elevated under the environment of high levels of T (10 and 20 ng/ml),thus increasing the conversion of T to E 2.Meanwhile,higher concentration of insulin suppressed the conversion of T to DHT through down-regulation of 5RD5A1 mRNA and protein level.The double effects lead to abnormal accumulation of testosterone.Conclusions:High level of insulin defects T transformation process by affecting the aromatase and 5α-reductasse1.Our data offered a possible mechanism which interaction between hyperinsulinemia and hyperandrogenism exacerbates polycystic ovary syndrome (PCOS) condition.