【作者】 孟君；

【导师】 魏莎莉；

【作者基本信息】 重庆医科大学 ， 遗传学， 2008， 硕士

【摘要】 背景:胚泡着床是哺乳动物生殖过程中妊娠建立的第一步,也是决定妊娠成功与否的关键。近年来,由于遗传、环境污染等因素所造成的不孕不育的问题日益严重,但现阶段辅助生殖领域最大的难题即胚泡着床率低这一世界性的课题还未得到很好的解决,所以我们有必要对胚泡着床这一重要的生命环节做出相关研究。胚泡着床过程包括定位、黏附及侵入三个步骤,而成功的着床要求子宫内膜与胚泡同步化发育,使子宫内膜能够在胚泡“着床窗”开放期这一很短的敏感时期达到容受状态,从而使子宫内膜成为能够吸附胚泡的表面,并诱发子宫内膜基质细胞发生蜕膜化转变。子宫内膜容受状态的改变涉及到众多基因的调控以及错综复杂的调节因子网络,其中细胞增殖、凋亡等复杂的分子细胞事件对这一改变的顺利进行起着重要作用[1]。此外,近年来的大量研究还表明各个生殖过程均涉及到细胞的增殖与凋亡,故与细胞增殖、凋亡相关的基因也成了胚泡着床过程分子机制的研究热点。PCNA (Proliferating cell nuclear antigen,增殖细胞核抗原)是由Miyachi等于1978年从系统性红斑狼疮患者的血清中发现的,因其存在于增殖性细胞中而得名,最初仅作为一种内源性标志物判断细胞增殖情况,近年来大量的基础研究表明PCNA具有众多复杂的生物学功能,在细胞增殖、凋亡及细胞周期调控等分子细胞事件中发挥重要作用[2]。Jeff R等采用基因芯片和原位杂交技术研究发现围着床期子宫内膜PCNA属于一种表达上调的基因,并推测其参与了子宫内膜增殖过程,并在在胚泡黏附及侵入过程中参与了子宫内膜的重建[3]。此外,PCNA的表达还与肿瘤的侵袭性转移密切相关[4],而胚泡着床与肿瘤侵袭性转移之间的生物学行为具有惊人的相似性[5],所以我们有理由怀疑PCNA可能参与了胚泡着床过程,而现阶段还尚无PCNA在胚泡着床过程中的作用的相关研究,故对这一课题进行研究是有意义的。目的:研究凋亡抑制基因PCNA在小鼠子宫内膜表达的规律性变化及其对胚泡植入的影响,初步探讨它在小鼠生殖中的作用。方法:1.收集未孕和早孕2、3、4、5、6、7天小鼠子宫内膜,共7组,每组20只小鼠。(1)运用实时荧光定量聚合酶链反应(FQ-PCR)技术测定PCNA mRNA的表达规律;(2)免疫组织化学SP法检测小鼠子宫内膜中PCNA蛋白的表达情况;2.于孕3天晚8时小鼠子宫角注射PCNA单克隆抗体,于孕8天观察着床胚泡数。结果:1. FQ-PCR:未孕及早孕各期小鼠子宫内膜组织均有PCNA mRNA表达,其中d2、d7组的表达量与未孕组相比无显著性差异,其余各组均大于未孕组,且在孕d4、d5达到高峰。2.免疫组织化学:染成棕黄色的PCNA蛋白表达于各组子宫内膜细胞胞核。d0至d5组基质细胞的阳性表达随着妊娠天数的增加呈现逐渐增强的趋势,d6、d7组逐渐降低;腺上皮细胞的阳性表达主要在d0、d3、d6组;腔上皮细胞的阳性表达主要在d3、d6、d7组。各组PCNA蛋白阳性细胞总数量表达规律与mRNA结果基本一致。3.于孕3天晚8时子宫角注射PCNA单克隆抗体,观察结果显示注射PCNA单克隆抗体组着床胚泡数明显低于对照组,具有显著性差异(P<0.05)。结论:PCNA在“着床窗”开放期高表达,说明PCNA可能通过某种途径启动细胞增殖凋亡过程,使子宫内膜发生容受性改变,保证胚泡在该期顺利着床,并为蜕膜化转变做准备。用PCNA单克隆抗体在蛋白水平阻断其生理功能,发现着床胚泡数明显减少,推测其很可能在胚泡着床中起重要的作用。更多还原

【Abstract】 Backgroud:Blastocyst implantation is the first step of mammal’s reproduction which determines whether pregnancy will develop successfully. In recent years, genetic, environmental pollution and other factors induce more and more unpregnency. However, implantation rate of IVF-ET is low and still have not been overcame now. So it is necessary to do some researches to this important reproduction event of blastocyst implantation.Blastocyst implantation process involves apposition, adhesion and invasion. Successful implantation needs synchronic development of blastocyst and endometrium, which leads endometrium to be receptive in the period of“implantation window”, and the receptive endometrium becomes a adsorptive surface, it also induces stromal cell decidualization. Endometrial receptive changes include plenty of genic regulations and complicate regulatory factors, and cell proliferation, apoptosis and other complicate molecular events play important roles in the change. In addition, recent researches suggest that cell proliferation and apoptosis participate in every step of reproduction, so researches of these genes are popular now in blastocyst implantation.PCNA ( Proliferating cell nuclear antigen) was found in SLE serum in 1978. It is named for it exists in proliferating cells. At first it was only a inner marker to indicate cell proliferating. However, in recent years, plenty of researches showed PCNA possesses numerous of intricate biological functions, which acts important in cell proliferation, apoptosis and cell cycle regulation. Jeff R applied microarray and in-situ hybridization discovered that PCNA is one of up-regulate gene in endometrium during preimplantation, and presumed it participates endometrial proliferation, and may act in a concerted fashion for remodeling of the endometrium during blastocyst attachment and invasion. Researches also suggested that the expression of PCNA correlates to tumor invasion, and blastocyst implantation and tumor invasion have remarkable similar biological behavior. So we presume that PCNA may participate in blastocyst implantation process. However, the role of PCNA during blastocyst implantation have not been researched at present stage, so it is significant to do.Objective: To investigate the expression rule of PCNA in the mice’s endometrium and analysis it’s role in blastocyst implantation.1. Endometrium o f mice in the early phase of unpregnancy and pregnant d2, d3, d4, d5, d6, d7entered into the study. There were 7 groups, each group had 10 mice. PCNA mRNA and protein expression were detected in the endometria of mice by real-time fluorescence quantitative PCR (FQ-PCR) and immuno- histochemical technique respectively.2. The number of blastocyst implantation was registered by injecting PCNA monoclonal antibody in horn of uterus at 20:00 on the pregnant d3.Results:1. The PCNA mRNA expressed in every group. The PCNA/β-actin mRNA of d2 and d7 compared to d0 had no significant difference, other pregnant groups were all higher, and at pregnant d4 and d5 the expression reached the maximum.2. Immunohistochemical analysis showed PCNA protein expressed in nuclei of every group. Positive stromal-cell quantity showed gradual hoist trend as days past in d0 to d5 groups, d6 and d7 groups showed gradual descent trend. Positive glandular epithelium cells mainly existed in d0, d3, d6 groups, and Positive luminal epithelium cells mainly existed in d3, d6, d7 groups. The rule of positive cell quantity was similar to mRNA.3. The numbers of blastocyst in horn of uterus which was injected PCNA monoclonal antibody is fewer than that of saline injected.Conclusion: PCNA expressed strongly in“implantation window”suggested that it may participate in some pathway to initiate cell proliferation and apoptosis, which induced endometrial receptive change to ensure blastocyst implanting in this period and prepared for decidualization. PCNA monoclonal antibody was used to intercept the physiologic function. We found that the number of blastocyst implantation markedly decreased, and deduced that PCNA may act important in blastocyst implantation.更多还原