基于全基因组DNA甲基化探讨不孕症肾阴虚证的表观遗传学机制

Kidney Governing Reproduction Theory is a crucial element of Visceral Manifestation Theory in traditional Chinese medicine. From proteomics, genomics and key gene expression regulation network point of view, our first three National Natural Science Fund subject researches have found that follicular fluid proteomics, ovarian granulosa cells genomics and key gene expression regulation network in patients of Kidney-yin deficiency changed, Chinese medicine for tonify-Kidney can intervene these changes, and then improve the pregnancy outcome in patients with infertility due to Kidney-yin deficiency.Based on this point, this project is to explore the epigenetic mechanism of infertility with Kidney-yin deficiency syndrome with genome-wide DNA methylation technology and bioinformatics method.Starting with Kidney-yin deficiency and normal group of patients with IVF-ET granulosa cell, analysis of granulosa cells genome-wide DNA methylation map is done panoramically with MeDIP-Seq and BSP technology to build a ‘infertility with Kidney-yin deficiency syndrome - genome-wide DNA methylation’ research platform. Kidney-yin deficiency patients with IVF-ET are selected after having Chinese medicines for tonify-Kidney. The difference among expression on granulosa cell genome-wide DNA methylation on the day of oocyte retrieval is analyzed, and then variance of expression on DNA methylation within two groups is compared. By combination of disease and syndrome and syndrome verification with formulas ,the expectations of this project are looking for the targets of Chinese medicines for tonify-Kidney, applying to clinical diagnosis and treatment, seeking for theoretical breakthrough in Kidney Governing Reproduction epigenetics mechanism and objective of Kidney-yin deficiency syndrome.

“肾主生殖”理论是中医肾藏象学说的重要组成部分，我们前三项国家自然基金课题研究证实：肾阴虚不孕患者卵泡液蛋白质组学、卵巢颗粒细胞基因组学和关键基因表达调控网络发生改变，补肾中药可干预这些变化，进而改善肾阴虚不孕患者的妊娠结局。在此基础上，本项目以挖掘不孕症肾阴虚证的表观遗传学机制为目标，运用全基因组DNA甲基化技术结合生物信息学方法，以肾阴虚与非肾阴虚试管婴儿患者卵巢颗粒细胞为切入点，利用甲基化 DNA 免疫共沉淀测序和亚硫酸氢盐测序技术，对两组患者取卵日卵巢颗粒细胞全基因组DNA甲基化图谱进行全景式分析，搭建“不孕症肾阴虚证—全基因组DNA甲基化”的研究平台。选取肾阴虚行试管婴儿的患者，通过补肾中药干预后，比较两组基因DNA甲基化表达的改变，寻找补肾中药的作用靶点，病证结合，以方测证，以期应用于临床诊断和治疗，力争在肾阴虚证的客观化研究和“肾主生殖”理论的表观遗传学机制上获得突破。

目的：通过全基因组亚硫酸氢盐测序技术（WGBS）研究肾阴虚不孕患者和正常女性卵巢颗粒细胞全基因组DNA甲基化图谱及甲基化差异表达基因，并进行功能验证。.方法：（一）选取肾阴虚不孕及非肾阴虚不孕行辅助生殖治疗的女性各60例，分别为肾阴虚组及正常对照组。取卵日留取卵泡液，分离冻存颗粒细胞。观察两组患者临床结局。WGBS技术获得两组患者全基因组甲基化图谱，通过GO及KEGG分析，获得肾阴虚不孕的目标基因及通路。.（二）选取辨证属肾阴虚不孕拟行IVF治疗的女性120例，以随机数字表法分为治疗组与对照组。另选取非肾阴虚不孕拟行IVF治疗的女性60例为正常对照组。三组患者分别在IVF治疗前两个月经周期给予六味地黄颗粒或安慰剂治疗。观察治疗前后患者肾阴虚证候评分的变化及临床结局。RT-PCR技术验证三组患者卵巢颗粒细胞中候选差异基因mRNA表达水平。.结果: （一）临床数据分析显示，肾阴虚不孕患者2PN受精率(52.9% vs61.8%)、优质胚胎率(48.5% vs 59.5%)及临床妊娠率(39.7% vs 63.8%)明显低于正常对照组，差异具有统计学意义（P＜0.05）。六味地黄颗粒治疗后，患者2PN受精率(60.5% vs 53.2%)、优质胚胎率(59.2% vs 47.7%)及临床妊娠率(58.6% vs 37.9%)明显改善，差异具有统计学意义（P＜0.05）。.（二）测序结果显示，肾阴虚与非肾阴虚不孕患者卵巢颗粒细胞DNA甲基化存在显著差异，共发现7498个DMR。主要涉及133个生物学过程（生物调节、代谢过程及发育过程等），定位于55个细胞组分（细胞、细胞膜及细胞器等）；具有44个分子功能（连接、转运蛋白等）。主要参与69个通路（卵巢类固醇合成、雌激素合成、PI3K-Akt信号通路、MAPK信号通路及细胞连接等）。.（三）RT-PCR验证结果显示：肾阴虚不孕患者卵巢颗粒细胞mTOR、ERβmRNA水平明显低于正常对照组，六味地黄颗粒治疗能够逆转肾阴虚不孕患者mTOR、ERβmRNA的表达，这一结果与甲基化测序结果一致。.结论：卵巢颗粒细胞mTOR和ERβ基因异常甲基化可能是肾阴虚患者卵母细胞质量下降及肾阴虚证候出现的核心病机之一，两者作为肾阴虚证候生物标志物和补肾中药六味地黄颗粒的调控靶点，为肾主生殖理论的表观遗传学证候本质研究提供了依据。