补肾法对小鼠胚胎干细胞在体分化为类卵细胞mTORC1-KITL信号通路的影响

Premature ovarian failure can lead to female infertility and low levels of estrogen in the body, which have serious impacts on women's physical and mental health. Clinical studies have demonstrated that Kidney-tonifying therapy for treating inpremature ovarian failure is more safe and effective than hormone replacement therapy. Previous study found the therapy could induce the differentiation of stem cells into oocyte-like cells (OLCs) in vitro, and its mechanism might be related to the differentiation from partial stem cells into granule-like cells during the induction, associated with inercellular microenvironment alteration. Thus we hypothesized that the Kidney-tonifying therapy could promote the differentiation of stem cells into OLCs in microenvironment of ovary, through activating mTORC1-KITL signal pathway of granule-like cells in cumulus-oocyte complexes (COCs). We will mark mouse embryonic stem cells with fluorescent marker, which would be injected into the fresh ovaries, and then transplant them into the autograft renal capsule. Furthermore , we choose immunity fluorescence method, promoting OLCs maturation and fertilization in vitro ,real-time RT-PCR and Western blotting. Finally we will observe the differentiation of stem cells in the microenvironment of transplanted ovary, and to verify whether Kidney-tonifying therapy could induce differentiation of stem cell into granule-like cells and of COCs into functional OLCs. We wish to explore that the mechanism might be related to the activation of the mTORC1-KITL signaling pathway of granule-like cells.

卵巢早衰可致不孕及低雌激素状态，严重影响女性生理和心理健康。临床研究证实补肾法治疗卵巢早衰较激素替代治疗安全有效。我们参与的前期研究发现：补肾法可在体外诱导干细胞向类卵细胞分化，其机制与诱导过程中部分干细胞分化为类颗粒细胞，细胞微环境改变有关。本研究提出“补肾-微环境-类颗粒细胞-类卵细胞”假说：补肾法可促使体内移植卵巢微环境中的干细胞分化为类卵细胞，其机制与激活类卵丘-卵母细胞复合体中类颗粒细胞的mTORC1-KITL信号通路唤醒干细胞分化相关。拟将小鼠胚胎干细胞荧光标记后，注入新鲜离体卵巢，再移植到同体小鼠肾包膜下；采用免疫荧光法、体外促成熟及体外受精、real-time RT-PCR和蛋白质印迹法，观察在体移植卵巢微环境中的干细胞能否在补肾法诱导下分化出类颗粒细胞并促使类卵丘-卵母细胞复合体向具备功能的类卵细胞分化，探索其机制是否与激活类颗粒细胞的mTORC1-KITL信号通路相关。