【作者】 王健；

【导师】 左嘉客； 申庆祥； 刘承权；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 胚胎着床(又称胚胎植入)是哺乳动物和人类生殖生理特有的一个环节，也是关系到妊娠成功与否的关键步骤。着床的顺利进行有赖于胚胎和子宫内膜的同步发育，即胚胎发育至胚泡阶段并完成孵化过程，同时子宫也处于接受状态，从而使得活化状态的胚泡能在“着床窗口”极其有限的开放时间内，及时与处于接受状态的子宫内膜建立紧密联系。这一显得非常精致的协同发育过程受到来源于胚胎和母体子宫内膜双方的各种激素、生长因子、细胞因子、蛋白酶等激素类分子和非激素类分子的调控，但其确切的分子机制仍不是十分清楚。为了寻找新的与胚胎着床相关的非激素类分子，以更深入地了解胚胎着床的分子机制，并探寻进行生殖调控的新环节，本实验室以小鼠为动物模型，分别收集了着床当日(孕D5天)小鼠着床部位和非着床部位子宫组织，以及着床前(孕D4天)和着床当日(孕D5天)小鼠胚泡，然后分别应用cDNA差减杂交和DD-RT-PCR技术，筛选得到许多在小鼠胚泡着床当日(孕D5天)的着床部位和非着床部位之间，或着床前后的胚泡之间存在特异性表达差异的基因的EST片段。但因人力、物力的限制，我们仅对其中很小一部分EST进行了测序分析，鉴别到5个新的着床相关基因，并在克隆获得这些基因的全长cDNA后在GenBank进行了登录，它们分别是：E4BP4(AY061760)、RGS2(AF432916)、ISP2(AF442819)、EMO-1(AY238936)和EMO-2(AY372183)。经GenBank Blast分析和相关文献查询发现，E4BP4是一种受IL-3调节的细胞核转录因子，但未发现该基因与胚胎着床相关的研究报道；RGS2参与G蛋白信号转导途径的调节，与细胞内钙离子浓度的调节以及T细胞增殖有关，其在着床中的作用未见有报道；ISP2是最近新发现的一种子宫组织特异基因，其表达受孕激素调节，并在胚胎着床期间呈特异性高表达，但其在胚胎着床中的作用尚有待于阐明；EMO-1与哺乳动物Sec63基因有高度同源性，而Sec63参与多种蛋白前体分子的翻译后加工；EMO-2是新基因，与人假定蛋白MGC50372有一定同源性，因此没有该基因的研究报道。MNSFD是本实验室正在研究的另一个新发现的着床相关因子，它是一种由抑制性T细胞合成的具有非特异性免疫抑制作用的淋巴因子，因其能抑制IL-4的分泌和Th2细胞的产生，所以推测其可能与母胎间免疫耐受的形成有关，但有待于研究证实。因抗ISP2抗体和抗MNSFβ抗体尚未市场化，为了获得这些蛋白分子的特异性抗体，用于其功能的研究，分别表达和纯化了ISP2重组蛋白，以及MNSFβ-hCGβ和GST-MNSFβ融合蛋白，并进而以这些重组蛋白作为抗原，分别制备了抗ISP2抗体和抗MNSFβ抗体。与此同时，本实验室的其他研究人员也分别制备了抗EMO-1氨基末端肽段抗体(抗EMO-1NTP抗体)、抗EMO-1羧基末端肽段抗体(抗EMO-1CTP抗体)、抗EMO-2氨基末端肽段抗体(抗EMO-2NTP抗体)和抗EMO-1羧基末端肽段抗体(抗EMO-2CTP抗体)。这些抗体其后被用于免疫组化研究和体内抗体封闭实验。应用原位杂交或／和免疫组化技术，对E4BP4、RGS2、ISP2、EMO-1、EMO-2在胚胎着床期间小鼠子宫内膜中的表达模式进行了分析，发现这些基因在胚胎着床前、后的表达均存在特异性差异，但表达部位各有不同，而且EMO-2的高表达不依赖于胚胎的存在，用我们自己制备的抗ISP2抗体进行的免疫组化结果与文献报道的原位杂交结果也相一致。为了进一步了解ISP2、MNSFβ、EMO-1和EMO-2在胚胎着床中的作用，以及探讨这些着床相关因子是否能成为发展新型抗生育技术的潜在靶分子，我们在小鼠体内进行了抗体封闭实验，结果表明，抗ISP2抗体、抗MNSFp抗体、抗EMO-1抗体和抗EMO-2抗体都具有抑制小鼠胚胎着床的作用，并呈剂量反应关系。而本实验室此前应用反义核酸技术进行的体内、外实验结果表明，EMO-1和EMO-2的反义核酸对胚胎的着床也有明显的抑制作用。我们分别收集了经抗ISP2抗体和抗MNSFβ抗体处理过和未经抗体处理过的孕D5天小鼠子宫组织，然后通过免疫组化研究，观察抗ISP2抗体对小鼠子宫中uPA和MMP-9表达水平的影响，分析其抑制胚胎着床的可能机制；并应用基因芯片技术，初步筛选出一些抗MNSFβ抗体处理后在小鼠子宫组织表达上调和下调的基因。为了建立基因剔除小鼠模型的建模技术，为以后深入研究这些基因功能缺陷对胚胎着床的影响极其作用机制提供动物模型，我们建立了具生殖系嵌合能力的C57BL／6J小鼠胚胎干细胞系，可用于今后制备转基因动物和基因剔除动物模型。综合以上研究结果，提示ISP2、MNSFβ、EMO-1和EMO-2在小鼠胚胎着床过程中起着重要作用，并且是发展新型抗生育技术的潜在靶分子，应进一步深入探讨这些基因在着床中的作用机制。更多还原

【Abstract】 Embryo implantation is a critical step in reproductive process specifically owned by mammalian and human being, and is the most relevant limiting factor for successful pregnancy. In order to establish an active interaction between the implanting embryo and the maternal endometrium during a very limited timespan defined as the "implantation window", the embryo should synchronously develop to the blastocyst stage and escapte from the zona pellucida, when the endometrium becomes receptive. Such an exquisite coordination involves the regulation of hormones and various non-hormonal molecules produced by embryonic and maternal tissues, including growth factors, cytokines, proteinases.With a view to further understand the precise molecular mechanism underlying embryo implantation and to find out new targets for the development of novel fertility regulation techniques, our lab used the mouse as a model to identify the novel implantation-related genes and explored the roles these genes played in embryo implantation. The mouse uterine tissues of implantation sites and inter-implantation sites were respectively collected at the time of implantation （Day 5 of pregnancy）, and the pre- and post-implantation embryos were also collected on Day 4 and Day 5 of pregnancy. Subsequently, the techniques of cDNA subtracted hybridization and DD-RT-PCR were respectively applied to screen the novel factors involved in mouse embryo implantation process. A large number of EST fragments of genes differentially expressed between the implantation and inter-implantation sites, or between the pre- and post-implantation implantation embryos, were screened out. However, having been short in labor power and research funds, just a small part of the EST fragment pools were sequenced for the further analysis, and finally 5 novel implantation-related genes were identified. The full-length cDNA of these genes were successively cloned and registered in the GenBank. They are E4BP4（AY061760）, RGS2（AF432916）, ISP2（AF442819）, EMO-1 （AY238936） and EMO-2（AY372183）.The results of GenBank Blast searches and references review showed that, E4BP4 is an IL3-regulated nuclear transcript factor, and no report on E4BP4 related to implantation was found; RGS2 participates in the regulation of G-protein signaling pathway and is involved in the regulation of intercellular Ca²⁺ mobilization and T cell proliferation, but there was also no report on RGS2 related to implantation; ISP2 was a newly-identified mouse uterine specific gene, and its expression was regulated by progesterone and sharply up-regulated during the period of implantation, but the roles of ISP2 played in implantation was still unclear; EMO-1 was highly homologous to mammalian Sec63 gene which was reported to be involved in the post-translation modification of various proteins; EMO-2 was a new gene and partly homologous to the human hypothetical protein MGC50372, and no report on EMO-2 and its homologous gene was found. MNSFP was a previously identified novel implantation-related factor that needed to be further investigated in our lab, and is a lymphokine produced by suppressor T cell. As it was reported that MNSFβ could inhibit the immune response in an antigen-nonspecific suppressive manner by inhibiting the secretion of IL-4 and production of Th2 cells, MNSFβ was presumed to participate in the formation of immune tolerance between embryo and maternal endometrial tissues.As the anti-ISP2 antibody and anti-MNSFβ antibody were not commercial available, we therefore prepared the recombinant ISP2 protein, as well as fusion proteins MNSFβ-hCGβ and GST-MNSFβ, respectively, and immunized animals by using these expressed products as the antigen to respectively prepare the anti-ISP2 and anti-MNSFβ antibodies. Meanwhile, the antibodies respectively against the EM0-1NTP, EMO-1CTP, EMO-2NTP and EMO-2CTP were also prepared in our lab. All of these antibodies were used in the subsequent IHC study and in vivo loss of function experiments.The uterine expression patterns of E4BP4, RGS2, ISP2, EMO-1 and EMO-2 during the period of embryo implantation were respectively investigated by in-situ hybridization and/or IHC. The results showed that, the uterine expression of these 5 genes were up-regulated during the implantation process, but the expression localization of these gene were not the same. Particularly, the high expression level of EMO-2 was embryo-independent. Furthermore, the expression pattern of ISP2 protein was well consistent with the ISP2 mRNA as previously reported.In order to further investigate the roles of ISP2, MNSFβ, EMO-1 and EMO-2 played in embryo implantation process, and to explore whether or not the potential target（s） for the development of novel contraceptive could be found among these genes, the antibody-blocking experiments were performed in mice. The results showed that, the antibodies against ISP2, MNSFβ, EMO-1 and EMO-2 caused a dose-dependent inhibitory effects on mouse embryo implantation in vivo. Consistently, the previous antisense RNA-blocking experiments of EMO-1 and EMO-2 also showed the inhibitory effects on embryo implantation. D5 pregnant mouse uterine tissues treated and non-treated by ISP2-antibody or MNSFβ were respectively collected. IHC analysis was carried out to observe whether or not the expression of uPA and MMP-9 proteins would be effected by the treatment of anti-ISP2 antibody. cDNA array technique was used to identify the up- and down-regulated genes in MNSFβ-antibody treated uterine tissues.In order to develop the techniques related to the development of knockout mouse model, which could be used to deeply investigate the inhibitory effects and the underlying mechanism caused by the function-deficiency of these implantation-related genes, we established a germ-line transmission using C57BL/6J embryonic stem cell lines that can be applied to generate transgenic or gene knock-out mice.To sum up, it could be concluded that, ISP2, MNSFβ, EMO-1 and EMO-2 play the critical roles in mouse embryo implantation, and consequently are potential targets for the development of novel contraceptive product. However, the mechanisms and the potential interaction among these implantation-related factors are still needed to be further explored.更多还原