松果体素激活自噬促进人卵巢颗粒细胞存活的分子机制研究

不孕症患者未受精卵子的颗粒细胞凋亡较受精卵子明显增加,胚胎质量与颗粒细胞凋亡率成负相关。动物实验结果表明，松果体素可通过膜受体抑制卵巢颗粒细胞凋亡；诱导自噬能促进小鼠生殖细胞存活，促进胚胎发育。本课题组预实验结果表明，松果体素可激活仓鼠卵巢细胞自噬。我们推测：松果体素与其受体结合后通过mTOR信号通路激活人颗粒细胞自噬，抑制其凋亡，促进其存活和继续发育，改善卵子质量、利于胚胎发育。为验证此假说，本课题①采用原代细胞培养、免疫印迹和流式细胞术等在细胞水平明确松果体素可通过激活自噬抑制颗粒细胞凋亡、促进其存活；②采用RNA干扰（SiRNA）、实时定量-PCR和免疫印迹等技术在分子水平研究松果体素作用的机制─在其受体介导下通过mTOR信号通路激活人颗粒细胞自噬；③利用转基因鼠模型验证松果体素通过激活自噬促进颗粒细胞存活、发育的机制，为将松果体素应用于临床、改善辅助生殖技术治疗结局提供理论依据。

Previous studies indicated that granulosa cells from unfertilized oocytes of infertility patients showed a significantly higher incidence of apoptosis compared to those of fertilized oocytes, and embryo quality had a negative correlation with the incidence of granulosa cells apoptosis. The results of the animal experiment showed that melatonin suppresses apoptosis of granulosa cells via its receptors (MT1 and MT2). Induction of autophagy improves germ cells survival in the female murine, and facilitates embryos development. Our preliminary experimental results indicated that hamster ovarian cells autophagy can be induced by melatonin. Therefore，we speculate that “cultured human ovarian granulosa cell synthesize a certain amount of melatonin, and exogenous melatonin induces human granulosa cell autophagy through mTOR signaling pathways, inhibits human granulosa cell apoptosis, after binding to its receptor, and ultimately improves human granulosa cell survival, facilitates oocyte quality, and contributes to embryo development. ” To verify this hypothesis, firstly, at the cellular level, we investigate how melatonin prevents human granulosa cell apoptosis and ultimately promotes human granulosa cell survival by activating cell autophagy using primary cell culture、Western Blotting and Flow Cytometry. Additionally, at the molecular level, we explore how melatonin induces human granulosa cell autophagy through mTOR signaling pathway mediated by its receptor using SiRNA、Real-time PCR and Western Blotting. Lastly, we verify that melatonin improves human ovarian granulosa cell survival by activating cell autophagy using transgenic mouse model. This project aims to provide theoretical supports for clinical infertility application of melatonin and improvement of assisted reproductive technology (ART) outcome.