益气血法通过调节GDF9分泌抑制小鼠超排卵卵丘细胞凋亡的研究

Oocyte and cumulus cell complexes (COCs) is the basic function unit of ovary. Oocyte-secreted factors (OSFs) can regulate the function of cumulus cells (CCs), including maintaining the low apoptosis rate of CCs, and CCs directly affect the quality of oocyte and embryo. In the process of in vitro fertilization (IVF), controlled ovary hyperstimulation (COH) impacts oocyte developmental potential, which is one of the important factors leading to low implantation rate. How to improve the COH oocyte quality is still under study in western medicine. We have found that Chinese traditional medicine Yiqixue recipe can increase the embryos implantation rate in IVF cycles. Animal experiments demonstrated COH reduces the expression of growth differentiation factor 9 (GDF9) in mice ovaries. While Yiqixue recipe could promote GDF9 expression in COH mice ovaries, and could improve the developmental potential of oocytes, but the mechanism is still unclear. GDF9 is one of the most important OSFs. On the basis of existing research, we suppose that yiqixue recipe could inhibit the apoptosis of CCs by promoting oocyte-secreted GDF9 levels, so as to improve the quality of oocytes. To test the hypothesis, experiments of COH mice would be conducted both in vivo and in vitro. Futhermore, the possible signaling pathways of Yiqixue recipe on COCs would be verified which would provide theoretical evidence for clinical application of Chinese herbal medicine in IVF.

体外受精(IVF)过程中，超排卵（COH）对卵母细胞发育潜能的影响是导致低着床率的重要因素，西医对如何改善COH卵母细胞质量尚处研究之中，我们以往的临床观察发现中药益气血法可以提高IVF胚胎种植率，动物实验提示该法可促进小鼠卵巢生长分化因子9（GDF9）表达，并可改善卵母细胞发育潜能，但其作用机制仍不清楚。GDF9来源于卵母细胞，具有调节卵丘细胞（CCs）凋亡的作用，而CCs直接影响卵母细胞的质量。本课题在已有的研究基础上，推论中药益气血法通过促进卵母细胞分泌GDF9，旁分泌作用于CCs，维持CCs低凋亡率，从而改善卵母细胞质量，并以COH小鼠为研究对象通过在体观察与体外实验进行验证，将祖国传统医药应用于IVF临床难点，期望能找到改善COH卵母细胞质量及发育潜能的途径，从而改善IVF临床结局。同时对益气血法可能的信号通路进行探讨，为其广泛的临床应用提供理论基础。

控制性超排卵（COH）是体外受精-胚胎移植（IVF-ET）周期的重要组成部分，已有研究证实COH可能破坏卵细胞中染色体的结构，影响染色体的功能，导致细胞中某些基因的表达缺陷，降低胚胎的质量和其着床能力。中医药介入IVF-ET周期收效显著，我们前期研究证实益气血补肝肾中药能提高胚胎移植成功率和妊娠率。本文主要观察益气血法经后增殖方对COH大鼠卵巢GDF9和Bim分泌的影响，探讨经后增殖方在抑制COH卵巢卵丘细胞（CCs）凋亡中的作用机制，并对其可能的信号通路进行验证。结果认为：①益气血法经后增殖方能通过促进COH大鼠卵巢卵丘-卵母细胞复合体（COCs）中卵母细胞分泌GDF9，而GDF9通过旁分泌作用周围的CCs，维持CCs的低凋亡率而改善COCs的功能，从而提高卵细胞质量；②益气血法经后增殖方能通过多途径抑制COH大鼠卵巢COCs和CCs中Bim的表达水平，途径之一是通过促进GDF9的分泌以维持Bim表达的低水平，从而抑制CCs凋亡；③益气血法经后增殖方对COH大鼠卵巢COCs和CCs中GDF9和Bim表达的调节是通过p38MAPK和NF-κB信号转导通路介导的；④经后增殖方含药血清作用于体外培养的超排卵模型大鼠卵巢颗粒细胞的最佳作用时间是24小时，最近作用浓度是3倍临床等效剂量。.本研究首次从细胞凋亡的角度探讨经后增殖方对CCs及胚胎质量的影响；填补了关于经后增殖方作用机理研究中动物体外细胞实验研究的空白，并获得该方含药血清的最佳作用浓度和时间；从分子水平初步探索了经后增殖方抑制CCs凋亡可能的信号通路，阐明中医药改善IVF-ET中卵母细胞质量及发育潜能的途径，为中医药在临床IVF-ET周期中的应用提供分子理论基础，进一步提高IVF-ET临床胚胎种植率与妊娠率，促进IVF-ET技术的进步。