【作者】 张丹；

【导师】 张炜；

【作者基本信息】 复旦大学 ， 妇产科学， 2012， 博士

【摘要】 子宫内膜接受胚胎着床的能力称为子宫内膜容受性。人类子宫内膜只在月经周期的20-24天具备接受胚胎植入的能力,称为“子宫内膜容受窗期”。在这一时期,子宫内膜在下丘脑-垂体-卵巢轴的精确调控下发生了特殊的形态、生理和分子生物学的变化来易化胚胎植入。子宫内膜容受性的建立是胚胎成功植入的决定因素,任何原因引起的子宫内膜容受性不能建立或建立延迟,都会导致胚胎着床失败。辅助生殖技术(ART)是解决人类不孕的重要手段,但试管婴儿技术(IVF)发明至今其成功率仍在20-30%徘徊。人们认为子宫内膜容受性缺陷大约占导致着床失败原因的2/3,而胚胎原因仅占1/3,无法诊断和治疗子宫内膜容受性异常也是ART成功的主要障碍。因此,容受性的诊断和评估对提高辅助生殖技术的成功率具有极其重要的临床意义。寻找子宫内膜容受窗期特异性表达的标志分子,阐明子宫内膜容受性形成的分子机制,将极大地推动生殖医学的进步。随着高通量筛选技术的发展,前期有许多研究应用基因组学及蛋白质组学技术来寻找子宫内膜容受性形成的标志分子。然而,由于样本量的限制、样本来源的不同、数据本身的高维度及分析方法和分析标准的差异,这些高通量筛选得出的结果并不一致。目前,可靠的子宫内膜容受性标志分子仍然没有找到,子宫内膜容受性的分子机制仍不清楚。重复高通量筛选无疑是一件既费钱又费力的事,而前期大量高通量筛选堆积了大量数据并没有得到充分利用。生物信息学的发展为我们有效利用前人的数据提供了平台,不仅可以避免浪费,而且从一定程度上扩大了样本量,增加了数据的可靠性。本研究的第一部分应用生物信息学的方法整合多中心数据进行分析,挖掘出在子宫内膜容受窗期特异表达的分子作为标志分子,并进一步验证部分分子在着床窗口期的表达规律,为寻找容受性标志分子及进一步研究子宫内膜容受形成的分子机制提供线索。胚胎着床过程包括胚胎在子宫内膜上定位、粘附与侵入等环节,该过程涉及细胞的增生、凋亡、粘附、侵袭和血管增生等细胞学行为。S100P是本课题在前期生物信息学挖掘中发现的在容受窗期子宫内膜特异性表达上调的分子。已知该分子广泛参与多种肿瘤的发生发展,与肿瘤细胞的增生、侵袭、转移、及血管生成有关,然而该分子与子宫内膜容受性形成的关系及在胚胎着床过程中的作用及机制目前还鲜有报道。本研究第二部分进一步观察S100P在子宫内膜和胎盘中的时空表达规律、P激素调节,并用RNA干扰和过表达技术探讨S100P在子宫内膜容受性建立中的作用及可能机制。本研究将有助于我们更好的理解胚胎植入伊始子宫内膜容受性建立的分子机制。第一部分子宫内膜容受性标志分子的生物信息学挖掘与分析目的：1、从现有的基因芯片数据库中挖掘子宫内膜容受窗期特异表达的分子,为寻找子宫内膜容受窗期标志分子提供线索。2、运用功能分析及相互作用网络分析方法,分析这些差异表达基因的功能及其相互作用网络,以进一步了解子宫内膜容受形成的分子机制。3、收集临床标本验证生物信息学挖掘的结果。材料与方法：1、应用生物信息学软件GeneSifter从现有的基因芯片数据库中挖掘筛选在子宫内膜容受窗期特异性表达的基因。2、用IPA软件分析这些差异表达基因的功能及相互作用网络。3、收集育龄妇女不同时期子宫内膜,用Realtime PCR验证这些差异表达基因在不同时期子宫内膜中的表达规律。结果：1、用生物信息学的方法整合已有的基因芯片数据,挖掘出子宫内膜容受窗期差异表达大于2倍的基因1543个,大于5倍的基因148个,大于10倍的基因31个。2、基因功能分析发现差异基因聚集最多的两个功能类是免疫反应和细胞周期。分子网络分析发现高度差异表达的基因主要组成了两个网络,一个与心血管系统的发育和功能相关,另一个与肿瘤的发生发展相关,其中TGFB1, TNF, ERBB2和FSH, CTNNB1,ESR1分别处于两个网络的中心,具有关键调节作用。3、我们选择了9个基因(DKK1、S100P、FAM148A、MACA、SFRP4、CXCL14、 CRISP3、ANXA4和SFN)用Realtime PCR进行验证,所有基因表达与生物信息学软件挖掘的结果一致。结论：用生物信息学的方法从已有的数据库中挖掘信息是一种可行的方法,不仅节约了资源,而且扩大了样本量,可以得到更加可靠的信息。第二部分s100P在子宫内膜容受性形成中的作用和机制研究目的：1、研究S100P在子宫内膜和胎盘中的时序表达规律。2、观察生殖激素对S100P表达的调节。3、探讨S100P对子宫内膜上皮细胞及基质细胞生理功能的影响。4、研究S100P影响子宫内膜细胞生理变化的信号机制。材料与方法：1、收集育龄妇女不同时期子宫内膜和不同孕期妇女的胎盘组织,用Realtime PCR、Western blot和免疫荧光研究S100P的表达规律。并分离培养原代人子宫内膜上皮细胞和基质细胞,用细胞免疫荧光检测S100P在细胞内的定位。2、分离培养原代人子宫内膜基质细胞和上皮细胞,培养子宫内膜上皮细胞系RL952和Ishikawa,培养人胚胎滋养细胞系Bewo。加入不同浓度雌二醇(E2)、孕酮(P4)和HCG培养,设空白对照,培养24-72h,用Realtime PCR和In-cell Western分析S100P表达的变化。3、体外培养原代人子宫内膜基质细胞和上皮细胞系RL952,应用慢病毒系统建立S100P干扰和/或过表达的稳转细胞株,检测S100P对细胞迁移和侵袭的影响。4、建立Bewo细胞球体与RL952和基质细胞的共培养体系模拟体外胚胎植入模型,观察S100P对胚胎与子宫内膜上皮细胞的粘附及胚胎滋养细胞在子宫内膜基质细胞中侵袭的影响。5、鬼笔环肽染色观察S100P对细胞骨架的影响；应用免疫荧光和Realtime PCR检测S100P对β-catenin信号通路和NFkB信号通路分子的影响。结果：1、S100P在分泌中期子宫内膜中特异性高表达,较另外三个时期高100多倍,在子宫内膜腺上皮和基质中表达均增高。在妊娠期中,S100P在早孕绒毛和足月胎盘中高表达,而在早孕蜕膜和中孕胎盘中表达较低。细胞免疫荧光显示S100P在胞浆和胞核中均有表达,其中以胞浆为主。2、与对照组相比,孕激素、雌孕激素联合和HCG可以上调原代人子宫内膜上皮细胞和基质细胞,及上皮细胞系Ishikawa中S100P的表达。在原代人子宫内膜基质细胞中,S100P对雌、孕激素和HCG的反应呈时间、剂量依赖性。3、在基质细胞中过表达S100P后,S100PmRNA水平上调5倍以上,免疫荧光显示蛋白表达水平也明显上调。在RL952和Ishikawa细胞中干扰S100P,干扰效率达98%以上。在子宫内膜细胞中,S100P抑制了细胞的迁移,但促进了细胞侵袭。S100P促进了Bewo细胞球在基质细胞中的扩展。在RL952细胞中,S100P干扰后,Bewo细胞球与RL952的粘附能力减弱。4、在子宫内膜上皮细胞中,S100P的表达变化与Ezrin的表达变化相反,干扰S100P促进了细胞骨架肌动蛋白在细胞膜的聚集。S100P抑制了子宫内膜基质细胞中β-catenin的核转位,而促进了NFkB的核转位,并且上调了NFkB信号通路的激活分子IKKB的表达。结论：1、S100P在子宫内膜容受窗期特异性高表达,参与了子宫内膜容受形成,可能在子宫内膜容受性的形成中起着重要作用。2、在子宫内膜细胞中S100P的表达受到E、P和HCG的精确调控。3、S100P影响子宫内膜细胞的迁移和侵袭,并且影响了胚胎在子宫内膜上皮细胞上的粘附和在基质细胞中的扩展。4、S100P通过调节细胞骨架重构、调节β-catenin和NFkB信号通路活性,影响了子宫内膜细胞的运动与侵袭,进而调节胚胎在子宫内膜上的粘附与侵入。更多还原

【Abstract】 Embryo implantation, as the most relevant limiting factor for successful pregnancy, is a critical step in pregnancy. The formation of endometrial receptivity is requisite for successful implantation. Physiologically, the human endometrium is receptive on the19-24th day of a menstrual cycle. This transitory period is called "implantation window", during which the endometrium undergoes extensive morphological and physiological changes to facilitate implantation of the embryo under the accurate regulation of the hypothalamic-pituitary-ovarian (H-P-O) axis. As one of the indispensable factor, the endometrial receptivity composes the major factor limiting assisted reproductive technologies (ART) and unexplained infertility. Therefore, the diagnosis and evaluation of endometrial receptivity has very important clinical significance, whitch will greatly promote the progress of the reproductive medicine.The past two decades have witnessed an explosive growth in the amount of genomic studies, which resulted in unprecedented amount of data accumulated. The same was true in the studies of endometrial receptivity. However, due to the nature of the data, variability, small sample volume and high dimension size, the results of high-throughput data were unreliable. Up to now, the reliable diagnostic markers are still lacking, the molecular mechanism under the endometrial receptivity remains unclear and the mass data are far from being fully used. The emergence of bioinformatics gives us the possibility to deal with this problem. It can not only avoid redundancy and unnecessary waste but also acquire more reliable results. We employed bioinformatics tools integrating microarray data related to endometrium receptivity from multi-center, mining potential biomarkers and further deciphering the functions and interactions of these molecules. Our studies provided a lot of valuable clues for further studies.Embryo implantation is a complex process that involves embryo apposition and attachment to the maternal endometrial epithelium, traversing adjacent cells of the epithelial lining and invasion into the endometrial stroma. There are many biological processes similar to tumors, such as cell proliferation, apoptosis, adhesion, invasion and angiogenesis. S100P, a small molecular calcium binding protein highly expressed in various tumor cells, participated in the proliferation, survival, metastasis and invasion of tumor cells, and related to the development, prognosis, and drug resistance of tumors, has been selected out specially highly up-regulated in the window of implantation in human endometrium in our previous works. It prompts that S100P may play a role in embryo implantation, however few publications focus on the role and mechanism of S100P in embryo implantation. We applied knock-down and overexpression to study the role and mechanism of S100P in the formation of endometrial receptivity, whitch will help us better understanding the mechanisms of human embryo implantation.Part One Data mining of genes related to endometrial receptivityPurpose:1. To mine potential biomarkers of the receptive endometrium by bioinformatics tools.2. Analyzing the functions and interactive networks of the potential biomarkers to decipher the mechanism of endometrial receptivity.3. Using clinical samples to substantiate the results of data mining.Materials and methods:1. CEL files were collected from public microarray data repositories and then uploaded onto GeneSifter to mining potential biomarkers whichspatial-temporally expressed in the mid-secretory phase of endometrium.2. The differentially expressed genes were then uploaded onto IPA for function and interactive network analysis.3. Endometrial samples from normal fertile women were collected and then realtime PCR was employed to verify the expression pattern of selected genes through the menstrual cycle.Results:1.1543gene sets were found to express differentially, of which148highly regulated genes were listed as potential biomarkers of the receptive endometrium.2. The function and pathway analysis identified the differentially expressed genes primarily involved in the immune response and cell cycle. Two networks related to the cardiovascular system and cancers were generated within the genes which changed more than10-fold. 3. Nine genes (DKK1, S100P, FAM148A, MAOA, SFRP4, CXCL14, CRISP3, ANXA4and SFN) were validated by real-time PCR.Conclution:It was a meaningful exploration of the existing data to acquire useful and reliable information, and our results undoubtedly provided valuable clues for further studies. Part Two The role and mechanism of S100P in the formation of endometrial receptivityPurpose:1. To examine the expression pattern of S100P in endometrium through the menstrual cycle and in placenta through the pregnancy.2. To investigate the hormonal regulation of S100P.3. To study the biological function of S100P in the establishment of endometrial receptivity and embryo implantation.4. To probe the potential molecular mechanism of S100P in the establishment of endometrial receptivity and embryo implantation.Materials and methods:1. Endometrium samples of different menstrual cycle were collected from normal fertile women and placental tissues were collected from nomal pregnant women. Realtime PCR, western blot and immunofluorescence were used to evaluate the expression and location of S100P.2. In vitro cultured endometrial cells, including primary endometrial stromal cells, epithelial cells and cell lines RL952and Ishikawa, were treated with various concentrations of17-beta-estradiol (E2), progenterone (P4) and HCG for different periods. Realtime PCR and in-cell western were employed to investigate the expression change of S100P.3. The expression of S100P was overexpressed in primary stromal cells and inhibited in endometrial cell line RL952and Ishikawa by lentivirus system. Cell migaration was detected by scratch test and cell invasionwas dctected by transwell assays.4. RL952or primary endometrial stromal cells-Bewo cell sphere co-culture model were established to investigate the effects of S100P to the adhesion of Bewo cell sphere in RL952cell monolayers or theinvasion of Bewo cell sphere in primary endometrial stromal cell monolayers.5. Phalloidin staining was used to observe the reorganization of cytoskeleton, Realtime PCR and immunofluorescence were employed to study the activation of|3-catenin and NFkB signaling pathway after overexpressing or inhibiting S100P in endometrial cells.Results:1. S100P was spatial-temporally up-regulated in the endometrium of mid-secretory phase during the menstrual cycle. And in the process of pregnancy, S100P was highly expressed in placenta of late pregnancy and early gestation villi?2. The expression of S100P could be up-regulated by estrogen, progesterone and HCG in primary stromal and epithelial cells and Ishikawa cell lines. And the regulation S100P in primary stromal cells was displayed in a time-and concentration-depend way.3. Expression of S100P mRNA and protein in RL952and Ishikawa cells were strongly inhibited by over98%. Expression of S100P mRNA and protein in primary stromal cells were overexpressed more than5-fold. S100P inhibited the migaration of endometrial cells but promoted their invasion. The overexpression of S100P facilitated the invasion of Bewo sphere in ESCs, and the RNA interference of S100P inhibited the adhesion of Bewo sphere to RL952monoplayer.4. The interference of S100P in RL952promoted the F-actin gathered in cell membrane. And the overexpression of S100P in ESCs inhibited the translocation of β-catenin into nucleus, but promoted the translocation of NFkB into nucleus and up-regulated the mRNA expression of IKKB.Conclusions:1. S100P was highly up-regulated in the endometrium in both epithelium and stoma during the window of implantation.2. The expression of S100P in endometrial cells could be regulated by E2, P4and HCG.3. S100P promoted the invasion but inhibited the migaration of endometrial cells.4. S100P increased the adhesion rate of embryo to the monoplayer of RL952and facilitated the expansion of Bewo spheres in the monoplayer of ESCs.5. S100P might influence β-catenin and NFkB signaling pathway involved in the establishment of endometrial receptivity.更多还原