基于关键基因表达调控网络的肾主生殖理论研究

This project is to explore the scientific connotation of Kidney Governing Reproduction Theory, with in vitro fertilization-embryo transplantation as technology platform, starting with Kidney-Yin deficiency, Kidney-Yang deficiency, Kidney-Qi deficiency and normal group of patients with IVF-ET granulosa cell. On the basis of the five fund researches before, through the collection on the target gene and transcription factors relation data, together with early differentially expressed genes Identification, build key genes groups of Kidney deficiency. With GO function enrichment analysis and bioinformatics pathway enrichment analysis, using western blot and RT PCR and other complementary technologies, do panoramic analysis for groups of granular cell protein expression of key genes. Combining the differentially expressed proteins have been identified, build the key genes - channel - protein regulation network platform of Kidney-Yin deficiency /Kidney-Yang deficiency/Kidney-Qi deficiency syndrome. Identify and determine the key genes and their regulation pathways network that Kidney deficiency and the mechanism of Kidney-tonify Chinese medicines is closely related to. Help to find the molecular biology path of infertility of Kidney deficiency. Provide theoretical and experimental basis for scientific prediction and diagnosis of Kidney deficiency and explore the essence of syndrome, in order to apply to clinical diagnosis, treatment, prognoses analysis and drug development, seeking for developing Kidney Governing Reproductive theory in gene, molecular biology path and protein.

项目以挖掘"肾主生殖"理论的科学内涵为目标，以体外受精-胚胎移植为技术平台，以肾阴虚、肾阳虚、肾气虚及正常女性取卵日卵巢颗粒细胞为切入点，在前五项基金研究基础上，通过收集整理已有目标基因与转录因子关系数据，结合前期鉴定出的差异表达基因，构建各组肾虚证的关键基因谱。利用GO功能富集分析及生物信息学通路富集分析，借助western blot及RT PCR等多种互补技术，对各组颗粒细胞关键基因的蛋白表达进行全景式分析；结合前期已鉴定出的差异表达蛋白，构建肾阴虚、肾阳虚、肾气虚证的关键基因-通路-蛋白质调控网络平台；辨识并确定与肾虚证发生和补肾中药作用机理密切相关的关键基因及其调控通路网络；研发肾虚不孕的分子生物学路径，为科学预测和诊断肾虚证、探讨其证候本质提供理论和实验依据，以期应用于临床诊断、治疗、预后分析和药物研发，力争在基因、分子生物学路径、蛋白质等多层次丰富发展"肾主生殖"理论。

目的 构建肾阴虚、肾阳虚、肾气虚证的关键基因-通路-蛋白质调控网络平台；辨识并确定与肾虚证发生和补肾中药作用机理密切相关的关键基因及其调控通路网络，丰富“肾主生殖”理论。 .方法与结果：1、肾虚组和正常组比较，存在差异表达基因361个；肾虚治疗组与安慰剂组相比，在肾虚证候积分变化、取卵数、优质卵数、优质卵率、受精卵数、优质胚胎数、优质胚胎率及临床妊娠率上均存在差异，治疗组均明显优于对照组；肾虚治疗组与安慰的差异基因按照GO功能分类，主要涉及到参与细胞增殖、生物合成、细胞凋亡、抗细胞凋亡、防御反应、炎症应答、细胞因子活性等方面；按照KEGG分类涉及到多个信号转导通路，包括P53信号通路、MAPK信号通路、雌激素信号通路、TGF信号通路等。2.与安慰剂组相比, 二至天癸治疗组后肾气虚组患者卵巢颗粒细胞 MDM2、p53、p21 mRNA 及蛋白的表达均下降, 差异有显著性（P<0.05）；六味地黄颗粒治疗后肾阴虚组患者卵巢颗粒细胞ERK信号通路中的FSH、ERK、cyclinD2蛋白及基因表达明显增高（P<0.05）；肾阳虚治疗组Nrf2-ARE抗氧化信号通路Nrf2、NQO1、HMOX1蛋白及基因相对表达量比肾阳虚安慰剂组高，差异有统计学意义（P＜0.05）。.结论 1.不孕患者在IVF-ET治疗中，应用补肾中药，可明显改善其肾虚症状，提高优质卵率、优质胚胎率，进而改善临床妊娠率；2.补肾中药可能通过纠正了肾虚症不孕患者卵泡液颗粒细胞增殖、凋亡、氧化应激等信号通路的关键因子基因及蛋白表达的变化，利于卵子的发育与成熟。进一步从基因组学及网络调控角度丰富了肾主生殖理论的内涵。