Kisspeptin介导的MAPK通路在PCOS卵巢功能障碍的调控作用及机制研究

Polycystic ovarian syndrome is a complicated endocrinological metabolic disease. It manifests as ovarian dysfunction involved with multiple systems, which affects reproductive function deeply. It has been determined that the system of kisspeptin/GPR54 in hypothalamus has central regulatory effect on HPG axis. However, there are no studies about the influence of ovarian kisspeptin/GPR54 system on reproductive function until now. In our previous studies, we have found that the gene expression of kiss1 and kiss1r in follicles and granular cells and the level of key proteins such as ERK1/2 and p38K in MAPK signaling pathway were decreased obviously in PCOS rats. While, the mechanism of effects of MAPK signaling pathway mediated by kisspeptin/GPR54 system on ovarian function in PCOS need to be further investigated. Taking kisspeptin/GPR54 as the starting point, which is based on in vivo and in vitro experiments by using animal module, molecular biology, biochemistry, and proteomics technology, we will investigate the direct effects of kisspeptin/GPR54 system on ovarian physiology. We will clarify the regulatory mechanism of this system in follicular development and ovulatory dysfunction in PCOS. Thus, on this basis, new scientific evidence and therapeutic target for the clinical treatment and prevention of PCOS will be provided.

多囊卵巢综合征(PCOS)是以卵巢功能障碍为主并累及多个脏器和系统的复杂内分泌代谢疾病，严重影响生殖功能。已经证实下丘脑kisspeptin/GPR54系统对HPG轴起中枢调控作用，但目前还没有卵巢kisspeptin/GPR54系统对生殖功能的研究。我们发现PCOS大鼠卵泡和颗粒细胞kiss1和kiss1r基因表达下调，MAPK信号通路中ERK1/2和p38K等关键蛋白下降，但kisspeptin/GPR54系统介导MAPK信号通路在PCOS卵巢功能障碍的具体调控作用及机制尚无系统的研究。本课题拟运用动物实验学、分子生物学、生物化学、蛋白质组学等，通过体内和体外实验，研究kisspeptin/GPR54系统对卵巢生理功能的直接作用，明确该系统在卵巢发挥功能相关的关键蛋白和信号传导通路，阐明该系统在PCOS卵泡发育和排卵障碍中的作用及分子机制，为PCOS临床治疗和预防提供新的科学依据。

本研究中，我们构建了PCOS大鼠，并发现大鼠体重明显增加，动情周期紊乱，卵巢体重比高于对照组。PCOS大鼠卵巢囊状结构增加，囊状卵泡内颗粒细胞层数减少，黄体减少，卵泡闭锁增多。同时，我们发现PCOS大鼠卵巢AMH、FSHR、Cyp17a1基因mRNA水平较对照组升高；GDF9、StAR、Cyp19a1、kiss1基因mRNA较其余两组下降；而Cyp11a1、kiss1r基因mRNA水平无明显差异。与对照组相比PCOS组大鼠血清睾酮升高，雌二醇及孕酮下降，卵泡刺激素水平无明显差异，黄体生成素升高，黄体生成素与卵泡刺激素比升高。PCOS组大鼠卵巢kisspeptin蛋白表达高于对照组，GPR54蛋白表达无明显差异。PCOS 组卵泡以及颗粒细胞中MAPK信号通路中ERK1/2、p38K、MEK1/2 等关键蛋白的表达水平下调, 通过对MAPK的调控能够有效恢复PCOS大鼠的生殖能力；渗透泵植入后kisspeptin-10添加组大鼠发情周期及卵巢体重比值较PCOS组、生理盐水组明显改善；大鼠卵巢可见卵泡内颗粒细胞层数明显增多，黄体数增加可见。大鼠卵巢卵泡发育、排卵及性激素合成相关基因，以及MAPK通路相关基因较PCOS组及生理盐水组均有所改善。与正常对照组相比PCOS组大鼠血清性激素水平明显好转。本研究表明，PCOS大鼠模型的卵巢kisspeptin/GPR54系统表达下降。卵巢局部渗透泵注射kisspeptin能改善多囊卵巢大鼠发情周期紊乱情况，卵泡发育、排卵相关基因表达情况及血清性激素异常水平，同时能够提升MAPK信号通路中相关基因的表达量，提示在PCOS大鼠中，由于MAPK信号通路介导的卵巢kisspeptin/GPR54系统信号传递受损，导致卵巢中颗粒细胞凋亡增加，从而导致卵泡发育障碍和排卵异常。本次研究为PCOS临床治疗和预防提供了新的科学依据。并且我们还找到一种潜在的对PCOS大鼠的生殖功能有改善的药物TAK-448。