基于microRNA-29调控RNA去甲基化酶FTO介导m6A甲基化修饰探讨补肾促排卵汤治疗氧化应激致DOR的作用机制

育龄期女性卵巢储备功能下降（DOR）是不孕症研究领域热点难点问题。国医大师夏桂成运用补肾促排卵汤治疗DOR，临床疗效显著。前期研究证实补肾促排卵汤通过FSH-FOXO信号轴抑制颗粒细胞凋亡，保护氧化应激损伤。由于卵母细胞成熟过程中DNA转录活性被抑制，导致DOR患者颗粒细胞和卵母细胞DNA甲基化水平未发生显著变化，近期诸多证据证实m6A 参与调节卵母细胞发育。本项目借助人卵巢颗粒细胞、DOR小鼠模型和卵泡体外培养模型，以miRNA-29为靶点，结合外泌体和m6A测序，运用免疫印迹、RT-PCR等分子生物学技术，分析DOR患者外泌体miRNA和颗粒细胞m6A表达水平，筛选 miRNA与FTO相关作用蛋白，从体内外验证补肾促排卵汤通过miRNA-29调控FTO介导m6A修饰，通过FTO-YTHDF3-FOXO3抑制颗粒细胞凋亡，通过FTO-mTOR激活卵母细胞发育，最终改善卵子质量和妊娠结局。

Diminished ovarian reserve（DOR）in childbearing women is a hot and difficult issue in the field of infertility research. Professor Xia Guicheng applied the Tonifying kidney and Inducing Ovulation Decoction to the treatment of Diminished ovarian reserve, and the clinical effect was significant. Previous studies have confirmed that kidney-tonifying and ovulation stimulating decoction can inhibit granulosa cell apoptosis through FSH-FOXO signal pathway and protect against oxidative stress injury. However, due to the inhibition of DNA transcriptional activity during oocyte maturation, DNA methylation levels of granulosa cells and oocytes in DOR patients did not change significantly. Recent evidences have confirmed that m6A is involved in the regulation of oocyte development. In this study, We used Human granulosa cells, DOR mouse model t and antral follicle culture system to analyze the expression levels of miRNA in exosomes and m6A in granulosa cells of DOR patients and screen for miRNA and FTO-related proteins. The purpose of this study is to verify that kidney-tonifying and ovulation stimulating decoction can regulate FTO mediated m6A modification by microrna-29a, inhibit granulosa cell apoptosis by FTO-YTHDF3-FOXO3 signal pathway, activate oocyte development by FTO-mTOR signal pathway in vivo and in vitro, and ultimately improve oocyte quality and pregnancy outcome.