生长分化因子-8在多囊卵巢综合症患者颗粒细胞调控芳香化酶表达的作用及分子机制

Growth differentiation factor-8 is involved in multiple reproductive activities. Aromatase (AROM) is the enzyme responsible for conversion of androgens to estrogens. The abnormal expression of AROM will result in steroidogenesis disorder, such as the excessive level of androgen in Polycystic Ovary Syndrome (PCOS) patients. However, whether GDF-8 is involved in the regulation of AROM expression in granulosa cells from PCOS patients is totally unknown. Our preliminary results showed that the concentration of GDF-8 in the follicle fluid of PCOS patients is much higher than that in normal patients. Furthermore, treatment with GDF-8 up-regulated AROM expression in cultured granulosa cells from non-PCOS patients, while GDF-8 down-regulated AROM expression in PCOS patients. Accordingly, it is proposed that the inhibition of GDF-8 on AROM expression may lead to excessive androgen of PCOS patients. Furthermore, the molecular mechanism of GDF-8 in the regulation of aromatase expression in PCOS and non-PCOS patients is different. In the current study, by using granulosa cells from PCOS and non-PCOS patients, through inhibitor or siRNA techniques, we will clarify the function of growth differentiation factor-8 on the aromatase expression in granulosa cells derived from PCOS patients and the underlying molecular mechanism on different aspects, including receptors, signaling pathways and transcript factors. The results obtained from this proposed study will provide not only important insights into the molecular mechanisms mediating GDF-8-regulated aromatase expression in PCOS patients, but also a novel clinical target that could be used for the prevention and treatment of excessive androgen of PCOS.

生长分化因子8(GDF8)参与多种生殖活动的调节。芳香化酶(AROM)促进雄激素向雌激素转化，在多囊卵巢综合症(PCOS)患者的低表达可导致其高雄激素血症。但GDF8是否参与PCOS患者AROM表达的调控未见报道。我们前期研究发现行体外受精技术助孕的PCOS患者取卵日卵泡液中的GDF8浓度高于非PCOS患者，且GDF8在PCOS患者颗粒细胞抑制AROM表达，在非PCOS患者则促进AROM表达，据此我们提出假说：GDF8在PCOS患者的高表达抑制AROM产生，从而导致PCOS高雄激素血症；此外，GDF8在PCOS患者调控AROM表达的分子机制与非PCOS患者不同。本课题选取来源于PCOS和非PCOS患者的颗粒细胞，在受体、胞内信号通路及转录因子水平上使用信号通路抑制剂或小干扰RNA技术对比研究GDF8调控AROM的分子机制，为探索GDF8在预防和治疗PCOS高雄激素中的作用提供新的思路。

项目背景：生长分化因子-8(GDF-8)属于转化生长因子β 超家族，参与机体各项生理活动的调节，但在调控人生殖活动中的研究较少。芳香化酶(CYP19A1)是促进雄激素向雌激素转化的关键酶，其表达异常可能导致激素生成过程紊乱，比如多囊卵巢综合症(PCOS)患者表现出的高雄激素血症。我们前期研究发现GDF-8 促进人颗粒细胞中芳香化酶的表达，但其上调芳香化酶表达的机制尚未明了，本项目重点研究GDF-8在人颗粒细胞中调控芳香化酶表达的分子机制及在多囊卵巢综合症高雄激素发生中的作用.主要研究内容：本研究以人原代颗粒细胞为研究模型，分别观察不同时间点和剂量GDF-8处理颗粒细胞后AROM mRNA和蛋白表达水平的变化。为检测GDF-8对AROM的调控过程是否通过与ALK4/5受体结合，对人颗粒细胞给予ALK4/5的特异性受体抑制剂SB431542进行预处理，通过观察细胞中mRNA及蛋白水平的变化明确该抑制剂是否可抑制GDF-8对AROM的调控作用。为验证GDF-8是否通过Smad2和Smad3下游信号通路参与AROM的调控，使用Smad4 siRNA敲除颗粒细胞内源性Smad4 表达，再观察GDF-8对 AROM调控作用的改变。使用ELISA检测正常不孕症患者与PCOS患者卵泡液中GDF-8浓度。用相同条件的GDF-8作用于PCOS患者颗粒细胞，观察细胞中AROM mRNA和蛋白表达水平的改变，探究GDF-8在PCOS患者颗粒细胞中对AROM的调控作用。.重要结果和关键数据：GDF-8促进人颗粒细胞AROM表达，具有时间和剂量依赖性作用。GDF-8通过激活ALK4/5受体，活化 Smad2/3 下游信号通路促进AROM生成。PCOS患者卵泡液中GDF-8的浓度显著高于非PCOS患者。与正常患者来源颗粒细胞作用不同的是，GDF-8在PCOS患者颗粒细胞中明显抑制AROM mRNA和蛋白水平的表达。 .科学意义：.本首次探讨人颗粒细胞中GDF-8通过经典 Smads 信号传导通路调控AROM表达；首次揭示GDF-8在PCOS患者中高表达并抑制AROM的表达，可能与PCOS患者血清高雄激素发生有关，为PCOS发病机制的研究和分子靶向治疗提供新视角和思路。