基于SCF/c-kit/PI3K通路研究补肾调肝法在治疗排卵障碍性不孕中对雌性生殖干细胞的调控机制

Ovulation disorders are the main types of infertility and follicular dysplasia is a key factor, so promote follicle growth is an important method of treatmen. Replenishing the kidney and soothing the livers, treatment is the primary method of TCM to treat such kind of infertility.Yangjing Zhongyu Tang in Fuqingzhu Gynecology is the representatives prescriptions. Yangjing Zhongyu Tang，s clinical and animal experiments have confirmed that the treatment has to promote follicle growth.In 2004, "Nature" found the phenomenon of female germ cells proliferation, and first proposed the female germline stem cells (FGSCs) concept. FGSCs play an important role in the maintenance of oocytes and production of follicles.Stem cell factor (SCF) is an important factor to promote stem cell division, the receptors and downstream signaling pathway is believed to be the critical path of follicular growth. The research propose the hypothesis: Mechanism of the replenishing the kidney and soothing the livers, treatment to ovulatory dysfunction infertility is based on the proliferation of SCF and its receptor excitation signal pathway FGSCs to promote oocyte and follicle cell proliferation.The research intends to use fluorescent quantitative RT-PCR, western blot techniques, by analyzing changes in SCF/c-kit/PI3K related molecules and specific markers of ovarian FGSCs, to research the effect link of the replenishing the kidney and soothing the livers, Representatives prescription Yangjing Zhongyu Tang to ovulatory dysfunction infertility.

排卵障碍是不孕症的主要类型，卵泡发育不良是关键因素，促卵泡生长是治疗的重要手段。补肾调肝是中医治疗此类不孕的主要方法，《傅青主女科》养精种玉汤为代表方剂，经临床和动物实验证实具有促卵泡生长的作用。2004年《Nature》刊载论文发现雌性生殖细胞具有增殖现象，首次提出雌性生殖干细胞（FGSCs）概念。后续研究表明，FGSCs在维持卵母细胞和卵泡生成中起重要作用。干细胞因子（SCF）是促进干细胞分裂的重要物质，其受体及下游信号通路被认为是卵泡生长发育的关键路径。本研究提出假说：补肾调肝法治疗排卵障碍性不孕的作用机制为SCF及其受体激发的信号通路调控FGSCs增殖，从而促进卵母细胞增殖及卵泡成熟。本研究拟采用荧光定量RT-PCR、Western Blot等技术，通过分析SCF/c-kit/PI3K相关分子及卵巢FGSCs特异性标记物变化，研究补肾调肝法代表方养精种玉汤治疗排卵障碍性不孕的机制。

背景:排卵障碍性不孕中卵泡发育不良是关键因素，补肾调肝法代表方养精种玉汤经临床和动物实验证实具有促进卵泡生长的作用。2004年首次提出雌性生殖干细胞（FGSCs）概念。FGSCs在维持卵母细胞和卵泡生成中起到重要作用。SCF其受体及下游信号通路被认为是卵泡生长发育的关键路径。研究内容:采用免疫荧光、RT-PCR、Western Blot、放射免疫、ELISE等技术，通过分析卵泡发育、性激素、FGSCs特异性标记物及SCF/c-kit/PI3K相关分子的变化，研究养精种玉汤促进卵泡发育、生殖干细胞增殖的作用及对SCF/c-kit/PI3K信号通路的影响。重要结果:中药可以通过促进卵母细胞增殖、卵泡发育、FGSCs的增殖，其作用机制与对SCF/c-kit/PI3K信号通路相关基因及蛋白表达调控有关。关键数据及意义:1.中药组小鼠卵巢中原始、初级、次级及优势卵泡数较模型组多，提示中药可以提高卵巢储备功能，促进卵泡的早、中、晚期发育。2.中药组GnRH、E2较模型组高，提示中药可以增强下丘脑GnRH及卵巢E2的分泌。3.中药组MVH、Brdu及Brdu-MVH双标记分布较模型组高，Brdu-MVH双标记高于中药+抑制剂组，提示中药可以增加卵巢内生殖细胞数量、提高卵巢内细胞的增殖能力，并增加FGSCs数量，PI3K信号途径的阻断减弱了中药作用。4.中药组Oct-4、MVH、Dazl、SCF、c-kit、PI3K基因mRNA表达均较模型组上调，PTEN、Foxo3a、p27基因mRNA表达下调，中药+抑制剂组、抑制剂组Oct-4、MVH、Dazl、SCF、c-kit、PI3K基因mRNA表达均下调，PTEN、Foxo3a、p27基因mRNA表达上调，上、下调程度抑制剂组＞中药+抑制剂组，提示中药可以提高Oct-4、MVH、Dazl、SCF、c-kit、PI3K基因的mRNA表达，降低PTEN、Foxo3a、p27基因mRNA表达，PI3K信号途径的阻断减弱了中药作用。5.中药组SCF、PI3K及pAkt的蛋白表达较模型组高，Foxo3a的蛋白表达下降，中药+抑制剂组、抑制剂组中SCF、pAkt的蛋白表达较中药组下降，下低程度抑制剂组＞中药+抑制剂组，提示中药可以提高卵巢组织中SCF、PI3K及pAkt的蛋白表，降低Foxo3a的蛋白表达，PI3K信号途径的阻断减弱了中药作用。