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

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

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。

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 5RD5A1were measured by quantitative realtime PCR after 4hours treatment of gradient insulin（1,10,100ng/ml）and T（1.25,2.5,5,10,20ng/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（E2）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（10and 20ng/ml）,thus increasing the conversion of T to E2.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.