分泌期uNK细胞及其相关因子高表达与胚胎反复种植失败的相关性研究

发布时间：2018-03-25 20:08

本文选题：反复种植失败　切入点：分泌期内膜　出处：《郑州大学》2017年硕士论文

【摘要】：研究背景反复种植失败(repeated implant failure,RIF)的具体分子机制尚不十分明确,目前已知2/3的RIF是由子宫内膜容受性失调引起的,究其原因可能与免疫相关,已有的免疫学研究表明,妊娠是一种同种异体排斥现象,正常妊娠的胚胎,为半同体组织抗原不被母体排斥影响而生存,蜕膜组织与正常免疫微环境有一定关联。子宫自然杀伤细胞(uterine natural killer cell,uNK)是子宫内膜特有的自然杀伤(NK)细胞,表面特异性标记是CD56,绝大部分uNK细胞表型为CD(56bright)CD16(-),细胞毒性低,分泌免疫调节细胞因子、血管生长因子等,在着床和蜕膜免疫调节中发挥积极功能作用。反复种植失败患者分泌期CD56+细胞比率高于正常妇女,提示uNK细胞可能在反复种植失败中对子宫内膜容受性的调节起到一定作用。白细胞介素-15(interleukelin-15,IL-15)是属于Th1相关性细胞因子,有研究显示它能促进NK细胞的分化和功能作用,正常妇女子宫内膜分泌期IL-15 mRNA表达上调,在分泌晚期升高为胚胎着床做好准备。IL-15是否在反复种植失败的患者分泌期内膜的表达增加,需要作进一步研究。有研究提示当肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)表达过高时,打破了种植窗Th1/Th2因子的平衡,抑制子宫内膜蜕膜化进程,促使上皮细胞凋亡,增加Th1型细胞因子的免疫杀伤反应,阻断滋养细胞的侵入,产生前列腺素和活化金属蛋白酶(MMPs)如MMP-2和MMP-9,降解绒毛的细胞外基质,引发早期妊娠失败。因此,uNK细胞与胚胎种植、胎儿生长发育和早期胎盘密切相关,这种关系一旦失去平衡,就会引起反复种植失败或早期自然流产。在各种打破免疫因素平衡的改变可能导致胚胎着床失败,因此本研究检测RIF子宫内膜CD56、IL-15、TNF-α的表达改变,探索其与RIF发生的相关性。目的1.分别检测RIF与正常分泌期子宫内膜CD56、IL-15、TNF-α的表达情况。2.探索CD56、IL-15、TNF-α在RIF患者子宫内膜分泌期是否表达相关,为RIF患者的临床治疗提供理论依据。材料与方法本研究课题经过郑州大学第一附属医院的伦理委员会批准,研究中所有入组研究对象均已告知实验内容并已签署知情同意书。选择2016年9月至2017年3月于本院生殖中心行IVF-ET/ICSI-ET/Thaw-ET助孕的患者中有助孕妊娠史拟再次助孕者为对照组,以至少移植4个优质胚胎或至少移植三个新鲜周期或冷冻周期后仍未能实现临床妊娠的患者为实验组。本实验分为两个部分,第一部分实验包括10例对照组和12例实验组,用qRT-PCR法检测子宫内膜CD56、IL-15、TNF-α的定量表达;第二部分实验包括10例对照组和12例实验组,用免疫组化法检测子宫内膜CD56、IL-15、TNF-α的定位表达。结果1.qRT-PCR法检测结果显示,CD56、IL-15、TNF-α在分泌期正常子宫内膜与反复种植失败患者内膜均有表达,但在RIF组表达均显著高于对照组,差异有显著统计学差异(CD56:4.533±2.616vs.1.302±1.163,P=0.002;IL-15:4.064±2.247vs.1.614±1.713,P=0.01;TNF-α:6.313±1.912vs.1.479±0.986,P=0.000)。2.免疫组化结果显示,CD56、IL-15、TNF-α在分泌期正常子宫内膜与反复种植失败患者子宫内膜腺上皮与基质细胞中均有表达,RIF组的平均光密度值显著高于对照组,差异有统计学意义(P0.05)。3.CD56的表达升高对RIF的诊断效能高,且与IL-15和TNF-α的表达均呈正相关(P0.05)。结论1.CD56、IL-15、TNF-α在反复种植失败分泌期子宫内膜比正常内膜表达均升高。2.CD56的表达升高对RIF有诊断价值,且与IL-15和TNF-α的表达可能存在一定联系,需进一步研究证实。
[Abstract]:The research background of repeated implantation failure (repeated implant failure, RIF) the specific molecular mechanism is not very clear, it is known that 2/3 RIF is caused by the imbalance of endometrial receptivity, the reason may be related to immune related immunological studies have indicated that pregnancy is an allograft rejection and normal pregnancy embryos. Half body tissue antigen by maternal rejection and survival, have a certain relationship with decidual tissue and normal immune microenvironment. Uterine natural killer cells (uterine natural killer cell, uNK) is a unique natural killer (NK) endometrial cells, surface specific marker is CD56, most of the uNK cell phenotype was CD (56bright) CD16 (-), low cytotoxicity, secretion of immunomodulatory cytokines, vascular endothelial growth factor, play a positive role in the function of implantation and decidual immune regulation. CD56+ secretion in patients with repeated implantation failure The percentage of cells was higher than that of normal women, suggesting that uNK cells may be repeated implantation failure to a certain role in the regulation of endometrial receptivity. Interleukin -15 (interleukelin-15, IL-15) belongs to Th1 related cytokines, studies have shown that it can promote the differentiation and function of NK cells, upregulation of the expression of IL-15 mRNA the normal endometrial secretion in late secretory increased expression of.IL-15 is good for embryo implantation in secretory endometrium in repeated implantation failure in patients with increased need for further research. Some studies suggest that when the tumor necrosis factor alpha (tumour alpha factor- alpha necrosis, TNF-) expression is too high, breaking the window of the implantation of Th1/Th2 factor the balance of inhibition of endometrial decidualization process, promote epithelial cell apoptosis, increase immune Th1 cytokine killing response, blocking the invasion of trophoblast cells, produce prostaglandin And the activation of metalloproteinases (MMPs) such as MMP-2 and MMP-9, the degradation of extracellular matrix of the villi, causing early pregnancy failure. Therefore, uNK cells and embryo implantation, fetal growth and early placenta are closely related, once the relationship is out of balance, it will cause repeated implantation failure or early spontaneous abortion. Break the immune factors of the change of the balance in may lead to embryo implantation failure, therefore the detection of endometrial RIF CD56, IL-15, change the expression of TNF- and explore its correlation with the occurrence of RIF. 1. RIF were detected with normal secretory phase endometrial CD56, IL-15, TNF- alpha.2. expression to explore CD56, IL-15, whether the period of expression secretion of TNF- in endometrium of patients with RIF, and provide a theoretical basis for the clinical treatment of RIF patients. The research materials and methods after the First Affiliated Hospital of Zhengzhou University ethics committee approved the study. All subjects were informed of experimental content and signed informed consent. From September 2016 to March 2017 in our hospital reproductive center IVF-ET/ICSI-ET/Thaw-ET help pregnant patients with pregnancy pregnancy history to help pregnant again for the control group, with at least 4 high-quality embryo transplantation Transplantation or at least three fresh or frozen cycle cycle after failed to achieve clinical pregnancy patients as the experimental group. The experiment is divided into two parts, the first part of the experiment including 10 cases in the control group and 12 cases in the experimental group, qRT-PCR was used to detect endometrial CD56, IL-15, quantitative expression of TNF- alpha; the second part of the experiment including 10 cases in the control group and 12 patients in the experimental group. The method of detection of endometrial CD56, with immuno IL-15, expression of TNF- alpha. The results of 1.qRT-PCR assay showed that CD56, IL-15, TNF- alpha in the secretory phase of normal endometrium and repeated implantation failure in patients with membrane were The expression, but the expression in RIF group were significantly higher than control group, there was significant difference statistically (CD56:4.533 + 2.616vs.1.302 + 1.163, P=0.002 + 2.247vs.1.614 + 1.713; IL-15:4.064, P=0.01; TNF-: alpha 6.313 + 1.912vs.1.479 + 0.986, P=0.000).2. immunohistochemistry results showed that CD56, IL-15, TNF- alpha in the secretory phase of normal uterus endometrial and repeated implantation failure were expressed in patients with endometrial epithelial and stromal cells, the average optical density of RIF group was significantly higher than the control group, the difference was statistically significant (P0.05) the increased expression of.3.CD56 on RIF expression and diagnosis efficiency is high, and IL-15 and TNF- alpha were positively correlated (P0.05) conclusion. 1.CD56, IL-15, TNF- alpha in repeated implantation failure in secretory endometrium than normal endometrial expression and increased the expression of.2.CD56 in diagnosing RIF, and the expression of IL-15 and TNF- and alpha may be related to the need to further research. This confirmed.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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