基于PI3K/AKT/mTOR 信号通路的补肾温阳祛风散寒法对肾阳虚型大鼠卵巢功能的保护机制研究

Full grasp of the domestic and foreign progress based on the latest research, this item would be selected PI3K/AKT/mTOR signaling pathway closely related cell proliferation and differentiation as the starting point.with the primary senile female kidney -Yang deficiency model rats as the research object and using the means of modern science and technology:the radiation immune method, enzyme league immune method, immunohistochemical method, immune fluorescence technology and retrovirus polymerase chain reaction method, this item would be researched tonifying kidney,wenYang,expelling wind and removing cold method of gengniantang inhibit the menopausal transition ovarian aging on PI3K/AKT/mTOR signaling pathway, and probe “the right amount expression of IGF-1 in perimenopausal follicle can inhibit PI3K/Akt/mTOR signal transduction pathway, lead to lower activity of ovarian function; Chinese medicine of tonifying kidney, wenYang,expelling wind and removing cold method can activate the fewer the pathway (by regulating the “the right amount expression of IGF-1”. this item would explore the relationship of IGF-1 and signal transduction pathway, explore the key scientific problems of gengni- antang inhibiting ovarian aging. The results are expected to provide ideas of molecular mechanisms of tonifying kidney, wenYang, expelling wind and removing cold method inhibiting the menopausal transition ovarian aging.Therefore, the present study is of significant scientific meaning and practical application value.

本项目在国内外最新研究进展的基础上，选择与细胞增殖和分化密切相关的PI3K/AKT/mTOR信号转导通路为切入点，以围绝经期肾阳虚大鼠为研究对象，采用放射免疫法、酶联免疫法、免疫组化法、免疫荧光技术及逆转录聚合酶链反应法等科技手段进行具有补肾温阳、祛风散寒法之经验方“更年汤”抑制围绝经期卵巢衰退的PI3K/AKT/mTOR信号通路的研究，并探讨“围绝经期卵泡中IGF-1的适量表达可抑制PI3K/Akt/mTOR信号转导通路的活性，从而导致卵巢功能低下；补肾温阳祛风散寒方药通过调节IGF-1在卵泡中的适量表达可激活该通路，促使卵巢功能恢复”。探索IGF-1与信号转导通路的关系，探讨更年汤抑制围绝经期肾阳虚大鼠卵巢功能衰退的关键科学问题，对阐明补肾温阳祛风散寒法抑制卵巢功能衰退的分子生物学机制，解决中药调控和延缓卵巢功能衰退的关键科学问题提供思路。具有重要科学意义和实际应用价值。

前期的实验及临床研究已证实温补肾阳祛风散寒法能明显改善 H-P-O 轴的各项激素水平，并开展了《温补肾阳祛风散寒法对肾阳虚型大鼠内分泌免疫网络调控及细胞凋亡相关因子的影响和作用机理的研究》的国家自然基金资助项目。在上述基础上，基于PI3K/AKT/mTOR信号通路，对“绝经前后诸症”治疗中温肾补阳，袪风散寒法进行深入研究，探究“更年汤”对绝经前后诸症的作用机理。本课题由在体动物实验及离体细胞培养两部分组成，第一部分包括造模、灌胃、取血、取出双侧卵巢备用；通过观察一般状况、各级卵泡计数、放射免疫法检测大鼠血清中 FSH、IGF-1；免疫组化法检测FSHR 、IGF-1R 卵巢组织表达；RT-PCR 法检测卵巢组织中 IGF-1mRNA、PI3KmRNA、p-AKTmRNA、p-mTORmRNA相对表达；免疫荧光技术检测卵巢组织中IGF-1、IGF-1R、PI3K、p-AKT、p-mTOR 的表达及在细胞中的定位量；经过统计学分析处理得出：①自拟更年汤对肾阳虚围绝经期模型大鼠的一般症状及体重有较好的改善作用。②自拟更年汤能够增加模型大鼠窦前卵泡及窦状卵泡数量。③自拟更年汤可能通过PI3K/AKT/mTOR信号通路调控卵泡发育，使卵巢功能得到一定改善。④自拟更年汤能上调肾阳虚围绝经期大鼠模型血清中IGF-I水平，使FSH水平降低；⑤自拟更年汤能上调模型大鼠卵巢组织上IGF-1、IGF-1R及FSHR蛋白因子在卵巢组织中的阳性表达。第二部分包括制备含药血清及灭活、筛选动情前期大鼠、卵巢颗粒细胞的分离与鉴定、用灭活后的含药血清培养卵巢颗粒细胞。通过流式细胞仪检测细胞周期；蛋白免疫印迹法检测卵巢颗粒细胞中PI3K、p-AKT、p-mTOR 蛋白的表达； RT-PCR 法检测卵巢颗粒细胞中PI3KmRNA、p-AKTmRNA、p-mTORmRNA 相对表达量。并得出：①更年汤含药血清能促进颗粒细胞从G1期向S期的转变；②更年汤含药血清在临床剂量上可提高PI3K的表达，但抑制Akt及其下游mTOR的表达；③本方一方面可促进细胞增殖，另一方面抑制细胞的凋亡。本实验通过研究补肾温阳祛风散寒法促卵泡发育的分子机制及该法对卵巢颗粒细胞信号转导通路（PI3K/Akt/mTOR）的调控作用，为中医理论的开拓性运用、中药替代性激素治疗、中医药现代化、中药新药的开发提供有效的依据。