补肾活血中药改善子宫内膜异位症卵泡微环境逆转卵泡生长障碍机制研究

Infertility rates of patients with endometriosis are as high as over 30%-50%. The mechanisms involved in the pathogenesis of endometriosis-associated infertility are closely related to impaired folliculogenesis and oocyte quality by follicular fluid. Do follicular fluid of endometriosis-associated infertility result in the granular cell dysfunction, or hinder nutrition and information communication between the granulosa cells and oocytes? The method of traditional Chinese medicine in treating endometriosis-associated infertility is to nifying kidney and activating blood. Clinical studies have shown that the herb medicines of kidney invigorate the circulation (neiyi fang) can significantly improve the pregnancy rate of patients with endometriosis. This project are to explore the key proteins resulting in infertility in follicular fluid of endometriosis-associated infertility (ME-FF) using mass spectrometry and protein chip analysis, ELISA verification; and to validate the toxic effects and clearly the targets and signaling pathways of the key proteins of ME - FF on granular cell function, oocyte maturation of rat ovarian follicles using the whole gene expression profile, according to the differences in gene expression analysis, including clustering analysis, GO, KEEG analysis. This project are still to clarify the mechanism of the herb medicines of kidney invigorate the circulation (neiyi fang) in treatment of infertility with endometriosis from the perspective of tissue microenvironment by in vitro and animal experiments.

子宫内膜异位症（Endometriosis，EM）不孕率高达30%-50%。目前认为EM卵泡液（EM-FF）影响卵母细胞成熟是导致不孕的重要原因。然而，卵泡液中成分复杂，是哪类或哪种物质发生了关键作用以及其具体机制目前尚不清楚。临床治疗EM性不孕症的常用方法是补肾活血。临床研究显示补肾活血中药（内异方）能够显著提高EM患者妊娠率。本项目拟采用质谱及蛋白芯片分析，ELISA验证，发掘EM不孕症FF（ME-FF）微环境致不孕关键蛋白分子；以鼠卵泡为实验对象，采用全基因表达谱研究，根据差异基因表达分析，包括聚类分析，GO分析、KEEG分析，验证ME-FF微环境致不孕关键蛋白分子对颗粒细胞功能、卵母细胞成熟的毒性作用，并明确ME-FF中致不孕关键蛋白分子影响卵泡发育的作用靶点及信号通路；通过体外及动物实验，从组织微环境角度阐明补肾活血中药（内异方）治疗EM性不孕症的分子机制，为临床应用奠定基础。

子宫内膜异位症（Endometriosis, EM）患者卵泡液异常微环境是导致EM性不孕症的重要因素。前期临床研究显示补肾活血中药（内异方）能够显著提高EM患者妊娠率。本研究采用iTRAQ及生物信息学方法对III/IV期EM性不孕症患者卵泡液致不孕关键蛋白进行筛选，通过扩大样本量采用ELISA验证，并检测卵泡液铁离子水平。结果显示，EM性不孕症患者卵泡液转铁蛋白水平显著降低，铁离子水平明显升高，其转铁蛋白饱和度为54.8%（对照组31.5%），超过45%，存在铁超载。进一步研究显示，III/IV期EM不孕症患者卵泡液可使小鼠卵母细胞活性氧水平明显增高，卵母细胞体外成熟率显著下降。当将III/IV期EM不孕症患者卵泡液转铁蛋白水平提高到正常，卵母细胞体外成熟率显著上升，但用转铁蛋白抗体阻断后卵母细胞体外成熟率再次下降。由此说明，转铁蛋白可能是III/IV期EM不孕症患者卵泡液微环境致不孕关键蛋白分子。内异方药物血清可降低EM性不孕症患者卵泡液处理后卵母细胞内氧化应激水平，改善EM性不孕症患者卵泡液对小鼠卵母细胞体外成熟的影响，提高卵母细胞体外成熟率。补肾活血中药还促进EM模型小鼠卵泡发育，改善其自然生殖能力。此外，本研究还发现内异方可抑制EM模型小鼠内异灶生长，减少盆腔粘连，改善盆腔微环境，改善各级卵泡发育，提高了EM小鼠的生殖能力，且无毒副作用，安全性好。本研究分别从卵泡液微环境及盆腔微环境角度阐明内异方治疗EM性不孕症的分子机制，为中医药治疗EM性不孕症奠定坚实的理论基础。