基于体外受精-胚胎移植卵巢颗粒细胞基因组学的肾主生殖理论研究

Kidney Governing Reproduction Theory is a crucial element of Visceral Manifestation Theory in traditional Chinese medicine. According to many National Natural Science Fund subject research, Chinese medicines for kidney-tonify is able to improve the quality of oocyte and endometrial receptivity. Based on this point, this project is to explore the scientific connotation of Kidney Governing Reproduction Theory with genomics technology and bioinformatics method. Starting with Kidney-Yin deficiency, Kidney-Yang deficiency, Kidney-Qi deficiency and normal group of patients with IVF-ET granulosa cell, analysis of granulosa cells genetic map regulation network is done panoramically with genomic chip technology and RT-PCR technology to build a 'kidney deficiency infertility-genomics' research platform. Through random parallel control study, Kidney deficiency patients with IVF-ET are selected after having Chinese Medicines for kidney-tonify. The difference among expression on granulosa cell genomics on the day of oocyte retrieval is analyzed, and then variance of expression on genes among every group is compared. The expectations of this project are looking for the targets of Chinese medicines for kidney-tonify, applying to clinical diagnosis, treatment, prognoses analysis and drug development, seeking for theoretical breakthrough in Kidney Governing Reproduction Genomics Mechanism.

"肾主生殖"理论是中医肾藏象学说的重要组成部分，我们既往多项国家自然基金课题研究结果表明补肾中药可提高卵细胞质量，改善子宫内膜容受性。在此基础上，本项目以挖掘"肾主生殖"理论的科学内涵为目标，运用基因组学技术结合生物信息学方法，以肾阴虚、肾阳虚、肾气虚与正常组试管婴儿患者卵巢颗粒细胞为切入点，利用全基因组芯片技术和RT-PCR技术，对各组颗粒细胞基因图谱表达调控网络进行全景式分析，搭建"肾虚不孕-基因组学"的研究平台。利用随机平行对照研究，选取肾虚行试管婴儿患者，通过补肾中药干预后，分析取卵日颗粒细胞基因组表达的差异变化，比较各组基因的表达差异，以期寻找补肾中药的作用靶点，以期应用于临床诊断、治疗、预后分析和药物开发，力争在"肾主生殖"的基因组学机制上获得理论突破。

目的 基于基因组学技术探讨肾虚引起的卵泡液颗粒细胞基因组学变化及补肾中药的干预作用，丰富“肾主生殖”理论。 .方法与结果：1、肾气虚组和正常组比较，存在差异表达基因395个；肾阴虚组与正常组比较，存在差异表达基因313条；肾阳虚组与正常组比较，存在差异表达基因319条。2、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。3、①肾气虚治疗组与安慰剂组比较，存在差异表达基因422条；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肾阴虚治疗组与安慰剂组比较，差异表达基因280条；按GO分析进行大体分类,主要涉及到细胞增殖的调节、细胞凋亡、免疫反应、防御反应、补体系统激活等方面。差异表达基因涉及的信号通路为：雌激素信号通路、细胞凋亡、补体系统。③肾阳虚治疗组与安慰剂组比较，共筛选到391条差异表达基因；与细胞增殖、凋亡有关的细胞周期调节蛋白,蛋白质泛素化，微管、微丝等细胞骨架的构建,线粒体及能量相关功能的因子，激素合成调节因子等；参与的通路是：能量代谢通路和TGF-β信号通路。.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基因表达的变化，利于卵子的发育与成熟。进一步从基因组学角度丰富了肾主生殖理论的内涵。