单个核细胞宫腔灌注改善子宫内膜容受性的免疫机制研究

In the application of assisted reproductive technology (ART), low implantation rate and repeated implantation failure (RIF) are the key problems. Low endometrial receptivity is the main reason for this problem. Successful embryo implantation is based on normal fetal-maternal crosstalk and immune response, and endometrial chronic inflammation could affect endometrial receptivity by interfering with normal immune response. Clinical observation showed that intrauterine autologous peripheral blood mononuclear cells (PBMC) administration may improve the endometrial receptivity and increase in vitro fertilization and embryo transfer (IVF-ET) successful rate, but the specific immunologic mechanism remains unclear. Presumably it is related with the change of inflammatory factors secreted by activited Th cell and NK cell in PBMC. This study is based on the successfully established model of endometrial injury and chronic inflammation of rabbit. In employing in vitro PBMC with endometrial cell co-culture and in vivo PBMC intrauterine administration, it tries to determine the key cell types and immunologic factors on both gene and protein levels, so as to explain how PBMC improves the endometrial receptivity in immunologic ways.

在辅助生育技术应用中，着床率低、反复着床失败是目前生殖医学专家面临的瓶颈问题，子宫内膜容受性差是其主要原因。胚胎成功着床伴随着母胎对话-免疫应答而实现，子宫内膜慢性炎症可通过干扰正常免疫应答反应而影响子宫内膜容受性。临床观察提示反复着床失败患者应用自体外周血单个核细胞（PBMC）于胚胎移植前行宫腔灌注，可能改善子宫内膜容受性而提高体外受精-胚胎移植（IVF-ET）成功率。但目前其调节慢性子宫内膜炎症的具体免疫机制尚不清楚, 推测可能与PBMC中T细胞及NK细胞分泌炎症因子活性改变相关。本研究以子宫内膜损伤及慢性炎症家兔模型及临床中反复着床失败患者为研究对象，利用在体PBMC宫腔灌注改善内膜容受性及体外PBMC与子宫内膜细胞共培养两种手段，进行效应观察的同时，筛选并验证起作用的关键细胞类型及基因，阐明相关分子机制及信号通路，从基因和蛋白水平回答PBMC改善子宫内膜容受性免疫机制。

在辅助生育技术应用中，着床率低、反复着床失败是目前生殖医学专家面临的瓶颈问题，子宫内膜容受性差是其主要原因。胚胎成功着床伴随着母胎对话-免疫应答而实现，子宫内膜慢性炎症可通过干扰正常免疫应答反应而影响子宫内膜容受性。外周血单个核细胞（PBMC）经hCG诱导组后，IL-8 mRNA表达在PBMC体外培养12h和24h内持续增高，培养至48h后，IL-8 mRNA表达相较于24h降低。但是，同一时间段hCG诱导组PBMC的IL-8mRNA表达显著高于未诱导组。说明hCG诱导促进了PBMC炎症因子IL-8 在mRNA水平上的表达。hCG诱导的Ishikawa细胞中LIF的表达水平明显高于hCG未处理组和空白对照组，E-cadherin明显低于hCG未处理组和空白对照组，αVβ3，VEGFA, ICAM 在与PBMC共培养的Ishikawa细胞中表达增高，但hCG诱导和未诱导的差异不明显。MUC1，COX2在三组中表达均未检测到差异。临床患者正常对照组、反复着床失败患者PBMC灌注组和未灌注组子宫内膜活检标本，检测LIF 和E-cadherin mRNA的表达存在差异。证明hCG活化的PBMC可以从免疫和炎症通路改善子宫内膜容受性。