脂肪来源的外泌体miR-18b经MAP3K1促进PCOS颗粒细胞凋亡的研究

Dysfunction of apoptosis is the main cause of the abnormal folliculogenesis observed in Polycystic Ovary Syndrome (PCOS). Earlier studies have identified that adipose tissue is involved in the metabolism by exosome. PCOS is a complex endocrine disorder with abdominal obesity and abnormal metabolism of adipose. However, little is known about the role of adipose tissue-derived exosome in granulosa cells. We used miRNA microarray to analyze the difference in exosomal miRNA content between follicular fluid of PCOS and healthy control, and selected miR-18b, which was significantly increased in PCOS for further study. We also found that adipose tissue-derived exosomal miR-18b in PCOS was also significantly increased, compared with the control. Furthermore, we found that exosomal miR-18b promoted granulosa cell apoptosis most likely by regulating MAP3K1 pathway. To further investigate the role of adipose tissue-derived exosomal miR-18b in granulosa cell apoptosis, we will use clinical samples，animal in vivo study, molecular and cellular function research to analyze the difference of exosomal miRNA expression profile in follicular fluid between PCOS and the control, to clarify the role of the adipose tissue-derived exosomal miR-18b in promotion of granulosa cell apoptosis and to reveal the mechanism of miR-18b promoting apoptosis. We believe our project reveal for the first time that the adipose tissue-derived exosomal miR-18b plays an important regulatory role in granulosa cell apoptosis, providing a new theoretical basis for understanding the mechanism of PCOS follicular development arrest and prevention targets.

颗粒细胞过度凋亡是造成多囊卵巢综合征（PCOS）患者缺乏高质量卵母细胞的重要原因。近年研究表明，脂肪细胞可通过外泌体传递信息，调控其他细胞的活动。PCOS患者常伴有腹部肥胖和脂肪代谢异常，然而，其脂肪来源的外泌体对颗粒细胞有何作用尚未见报道。我们预实验发现：PCOS患者卵泡液所含外泌体中miR-18b升高幅度最大；而离体培养的PCOS脂肪细胞分泌的外泌体miR-18b也显著升高；体外研究显示PCOS外泌体miR-18b促进颗粒细胞凋亡，机制很可能在于调控MAP3K1通路。本项目拟开展临床、动物、细胞和分子研究，分析PCOS卵泡液外泌体miRNA表达谱变化，揭示脂肪细胞分泌的外泌体miR-18b促进颗粒细胞凋亡的作用，并解析其促进凋亡的机制。预期首次揭示PCOS脂肪细胞的外泌体可调控颗粒细胞凋亡，为认识PCOS卵泡发育障碍的机制以及寻求防治靶点提供新的理论基础。

多囊卵巢综合征（Polycystic Ovary Syndrome，PCOS）是青春期和育龄期妇女常见的内分泌代谢异常疾病。我国女性中PCOS发病率约为5.6%，由PCOS带来的不孕症占到了无排卵性不孕症75%。PCOS患者卵巢中虽有较多小卵泡，但无高质量的成熟卵母细胞产生。颗粒细胞过度凋亡是造成多囊卵巢综合征（PCOS）患者缺乏高质量卵母细胞的重要原因。近年研究表明，脂肪组织除了储能外，还是体内重要的内分泌器官，可通过外泌体传递信息，调控远端组织的生物学功能。PCOS患者常伴有腹部肥胖和脂肪代谢异常，PCOS发病过程中，内脏脂肪往往比体脂增长更快。然而，其脂肪来源的外泌体对颗粒细胞有何作用尚未见报道。为了回答以上问题，本项目从临床、动物、细胞和分子四个层面开展研究，发现PCOS患者和正常人群的卵泡液外泌体在纯度，粒径和浓度上无明显差异；miR-18b在PCOS患者组中的卵泡液外泌体和颗粒细胞中的含量均明显增高；同时，利用大鼠前脂肪细胞，在体外证明外泌体miR-18b的可传递性，在动物层面，利用PKH67外泌体染色的方法观察外泌体进入颗粒细胞，在动物水平验证外泌体的可传递性；另外，利用DHEA法构建PCOS动物模型，从卵巢形态和体积，雌激素水平验证动物模型构建成功，并发现相比野生大鼠，PCOS大鼠颗粒细胞凋亡明显增加，且用GW4869处理，可减弱其凋亡情况。同时，外泌体miR-18b可以下调MAP3K1信号通路，促进凋亡作用。我们的研究首次将PCOS的两个重要症状稀发排卵和腹部肥胖，通过外泌体(细胞间信息传递的重要载体)联系起来，将为PCOS的研究提供新的思路，使PCOS的研究不再拘泥于单一症状，且首次揭示PCOS脂肪细胞的外泌体可调控颗粒细胞凋亡，为认识PCOS卵泡发育障碍的机制以及寻求防治靶点提供新的理论基础。同时，有助于更全面认识外泌体miRNA的功能，并进一步提升外泌体miRNA在PCOS临床应用中的价值。