颗粒细胞的生物节律失调在雄激素受体介导的多囊卵巢综合征卵泡发育障碍的机制研究

Normal circadian rhythm in ovarian granulosa cells (GCs) is required for steroid hormone synthesis and metabolism and follicular development. The disruption of circadian rhythm is closely associated with infertility such as ovulation dysfunction. Polycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility in women of childbearing age. However, its etiology is still largely unknown. A pilot study of our team found that disrupted circadian rhythm in GCs of PCOS women, implying a breakthrough for understanding of molecular mechanism of follicular dysfunction. With primary culture of GCs and real-time PCR, we will detect the change of expression rhythm of core clock genes, clock controlled genes and androgen receptor (Ar) gene which is candidate gene for PCOS pathogenesis. With overexpression assay and luciferase report system, we will clarify the effect of core clock genes on transcription regulation activity of AR. With co-IP, we will prove the direct interaction between core clock proteins and AR. With ELISA and ChIP, we will investigate the role of core clock protein in androgens synthesis and metabolism and its underlying mechanism. With androgen receptor element related assay, we will analyze the feedback regulation of core clock gene transcription of AR. Thus, from the point of view of circadian rhythm, the current study will provide theory basis for etiology and clinical prediction and intervention of PCOS.

卵巢颗粒细胞（GC）的正常生物节律是甾体激素合成代谢、卵泡发育的必要条件，其失调与排卵障碍等不孕症的高发密切相关。多囊卵巢综合征（PCOS）是育龄期女性无排卵性不孕的首要病因，其发病机制至今未明。本项目组前期调研发现，PCOS患者GC存在生物节律失调，为其卵泡障碍机制的解析提供了新突破口。本项目拟借助原代GC培养、real-time PCR，明确PCOS患者GC中多种核心生物钟基因、钟控基因和PCOS致病候选基因雄激素受体（Ar）表达节律性的变化；借助过表达、荧光素酶报告系统，阐明核心生物钟蛋白对AR转录调节活性的影响；借助co-IP，验证核心生物钟蛋白与AR蛋白间的直接作用；借助ELISA和ChIP，研究核心生物钟蛋白对GC雄激素合成代谢的影响及分子机制；借助雄激素反应元件相关实验，分析AR对核心生物钟基因表达的反馈调控作用。从生物节律层面，为PCOS的病因学理解与临床诊治提供理论依据。

卵巢颗粒细胞（GC）的正常生物节律是甾体激素合成代谢、卵泡发育的必要条件，其失调与排卵障碍等不孕症的高发密切相关。多囊卵巢综合征（PCOS）是育龄期女性无排卵性不孕的首要病因，其发病机制至今未明。本项目组前期调研发现，PCOS患者GC存在生物节律失调，为其卵泡障碍机制的解析提供了新突破口。本项目借助原代GC培养、real-time PCR，明确了PCOS患者GC中核心生物钟基因、钟控基因和PCOS致病候选基因雄激素受体（AR）表达节律性的变化；借助过表达、荧光素酶报告系统，阐明了核心生物钟蛋白对AR转录调节活性的影响；借助co-IP，验证了核心生物钟蛋白与AR蛋白间的直接作用；借助ELISA和ChIP，研究了核心生物钟蛋白对GC雄激素合成代谢的影响及分子机制；借助雄激素反应元件相关实验，分析了AR对核心生物钟基因表达的反馈调控作用。根据本项目研究结果提示，以核心生物钟蛋白PER1为代表的卵巢局部生物钟紊乱通过干扰颗粒细胞中AR转录调节活性的发挥，降调下游参与雄激素合成代谢的关键酶，造成局部高雄状态，从而促进卵泡发育障碍的发生。此部分研究结果，从生物节律层面，为PCOS的病因学理解与临床诊治提供了有力的理论依据，具有致病机制研究思路创新性和临床疾病防治策略的前瞻性。