PI3K/Akt/mTOR通路调控卵巢颗粒细胞增殖分化及分泌功能的研究

Polycystic ovary syndrome(PCOS) is the most frequent reproductive dysfunction in women, one of the most frequent characteristic is insulin resistance from women with PCOS. The PI3k/Akt/mTOR pathway represents one of the main pathways implicated in cell proliferation and metabolism, it is important that the pathway is also a fundamental signaling pathway for insulin physiological functions, Thus, the growth and proliferation and metabolism of ovarian granulosa cells (GC) through this pathway, have become an attractive area of the molecular mechanisms of ovulation failure. Here we provide the report that it imitates the oocyte maturation in vivo to add preovulatory follicular fluid, the nutrient molecules and oocyte-secreted growth factors into GC culture medium, as it provides the physiological simulation environment to help GC culture in vitro, basing on the insulin resistant mechanistic understanding it is conducted on a normal granulosa cells which were from ICSI patients，and human luteinized granulose cells from PCOS patients. we detect the contents of estradiol and progesterone and inhibin B of the cells supernatant from different time points, we investigated an imbalance between cells supernatant concentration and the homeostasis model assessment of insulin resistance, the hypothesis is investigated that insulin resistance plays a role in the pathogenesis of reproductive abnormalities in PCOS. In the pathway S6K1 is a key regulator of protein, the subsequent studies are performed to investigate the molecular mechanisms of proliferation and metabolism associated with PI3K/Akt/mTOR pathway by inhibitor of the mTOR (Rapamyein) or combinations，Lentiviral expression vector containing human S6k1 is pLenti-S6K1 ，and small interfering RNA is pLenti-S6K1-siAkt. GC were cultured and transfected by lentivirus vector, targeted at blocking activation and phosphorylation of S6K1 gene, and GC were transfected by pLenti-S6K1, targeted at activating the phosphorylation and translations of Akt and mTOR and S6K1 gene in control culture system and modified culture system. Our findings provide a better understanding of relationship and evidence on the transcriptional activation and phosphorylation or gene silencing via the pathway involving in ovarian granulosa cells with insulinemia and insulin resistance. There is need for further studies to elucidate the exact role of improving insulin response and up- or down-stream signaling action. It may provide a novel molecular targeted therapy for PCOS.

胰岛素抵抗是多囊卵巢综合征（PCOS）的主要特征之一，胰岛素抵抗通过PI3K/Akt/mTOR通路会影响细胞增殖分化和代谢。本研究拟揭示该通路影响下卵巢颗粒细胞增殖分化及内分泌功能与PCOS胰岛素抵抗、卵泡发育停止的关系。研究中利用优化的颗粒细胞培养条件，通过添加胰岛素生长因子和mTOR抑制剂研究对通路的影响，筛查正常的卵丘冠颗粒细胞和PCOS黄素化颗粒细胞中通路相关蛋白差异性表达，分析确定S6K1和IRS1之间的调控关系，在此基础上运用慢病毒过表达载体pLenti-S6K1及慢病毒干扰载体pLenti-S6K1-siRNA，分析S6K1基因在两种颗粒细胞中的调控功能及由此影响的颗粒细胞的增殖分化和分泌功能，探讨颗粒细胞增殖及分泌功能影响PCOS早期卵泡发育停止的机制，阐释PCOS胰岛素抵抗与颗粒细胞增殖及凋亡的相关性，为PCOS的诊治提供科学的研究依据，为PCOS的基因治疗提供新的靶点。

胰岛素抵抗通过PI3K/Akt/mTOR通路影响多囊卵巢综合征（PCOS）中颗粒细胞的增殖分化和代谢。通过本项目的实施揭示该通路影响下人卵巢颗粒细胞增殖分化相关因子p70S6K1、ERK1/2、caspase-3和FOXO3a及E2和P4的分泌都与PCOS胰岛素抵抗、卵泡发育停止有密切关系。项目研究中利用优化的颗粒细胞培养条件和培养设备，通过添加EGF、ITS、生长重组GDF-9因子和抑制剂ICI182 780等方法，筛查正常的卵丘冠颗粒细胞和PCOS黄素化颗粒细胞中通路相关蛋白差异性表达，证实颗粒细胞中S6K1和IRS1之间的调控关系，同时也证实了雌激素受体基因ERβ和FOXO3a基因在两种颗粒细胞中的调控功能，以及颗粒细胞旁分泌因子GDF-9对颗粒细胞增殖分化中ERK1/2和caspase-3的影响，同时利用颗粒细胞和卵母细胞共培养证实了PCOS颗粒细胞增殖对卵母细胞生长发育的影响。项目组也按照申请项目的评审专家意见建立了PCOS模型大鼠,体内实验证实颗粒细胞增殖及分化因子影响PCOS早期卵泡发育停止的机制，我们的研究阐释了PCOS胰岛素抵抗与颗粒细胞增殖及凋亡的相关性，为PCOS的诊治提供科学的研究依据。