【作者】 郗强；

【导师】 杨戎；

【作者基本信息】 重庆医科大学 ， 遗传学， 2010， 硕士

【摘要】 背景:胚胎着床是决定人类和哺乳动物妊娠成功与否的关键,成功着床取决于接受态子宫内膜与具侵入性胚泡间的同步协调作用。子宫内膜仅在某一特定时期允许胚胎植入,此时子宫内膜对胚泡具有容受性,这一短暂的时期称为“着床窗”。胚胎着床包括胚泡脱透明带、定位、粘附、侵入以及植入等几个阶段,这个过程涉及到很多生物因子的调控和参与。肿瘤坏死因子α转换酶(TACE/ADAM17)因其能水解膜型肿瘤坏死因子(TNFα)而得名[1]。它具有ADAM家族的共有序列:信号肽,前肽,金属蛋白酶区,解整合素区,富半胱氨酸区,跨膜区和胞浆区[2,3]。可水解多种膜蛋白的跨膜区域释放胞外区,从而激活膜蛋白使其具有生物活性。已有研究表明,TACE在多种肿瘤如:乳腺肿瘤、肾细胞癌、肝细胞癌、前列腺癌、胰腺癌、结肠癌中有高表达[4,5],高表达的TACE在肿瘤发生、发展过程中起着重要作用。目前认为胚胎着床过程与肿瘤侵袭转移过程非常相似,很多在肿瘤中起作用的生物因子同样也在胚胎着床过程中起作用。我们前期的研究也表明:TACE在动情周期及早孕小鼠的子宫内膜中存在规律性表达,未孕期表达明显低于孕期,在早孕期随着妊娠天数的增加其表达呈现逐渐增强的趋势,在妊娠第4天达到峰值[6],主要表达在子宫内膜上皮细胞中。故推测TACE参与了胚胎的着床过程。目的:本实验通过检测肿瘤坏死因子α转换酶(TACE)在小鼠子宫内膜上皮细胞中的表达及其在胚泡、上皮细胞共培养体系中对胚泡粘附的影响初步探讨TACE在小鼠生殖中的作用以及其与胚胎着床的关系。方法:1.体外培养孕4天(d4)小鼠子宫内膜上皮细胞和胚泡,构建胚泡体外着床模型;2.通过细胞免疫化学染色方法鉴定体外培养体系中小鼠子宫内膜上皮细胞;3.运用RT-PCR以及细胞免疫化学方法分别检测培养体系中TACE mRNA和蛋白的表达;4.向体外着床模型中添加不同浓度的TACE抗体,观察对胚泡体外着床的影响,并用细胞免疫化学法检测抗体干预后各培养体系中TACE蛋白的表达。结果:1.原代培养小叔子宫内膜上皮细胞生长旺盛。细胞鉴定结果显示:上皮细胞对角蛋白抗体有棕黄色的阳性表达,对波形蛋白抗体为阴性表达;2.PCR结果显示TACE mRNA在培养体系中有高表达,免疫化学的结果与RT-PCR结果相一致;3.小鼠胚泡体外共培养具有较高的粘附率,添加TACE抗体后胚泡帖附率均显著低于对照组帖附率(P<0.05),且随着抗体浓度的降低胚泡粘附率随之升高,免疫化学检测结果也显示随着抗体浓度的降低TACE的表达量随之升高。结论:TACE mRNA和蛋白在妊娠d4子宫内膜上皮细胞有高表达,这可能是通过诱导子宫内膜物质脱落,调整子宫内膜的内环境而适应胚胎的侵入。抗体干预实验显示添加TACE抗体后胚泡帖附率均显著下降,这提示TACE可能参与了胚泡着床过程,有利于小鼠胚胎粘附、扩展,为胚胎的着床做准备。更多还原

【Abstract】 Background:Blastocyst implantation is one of the earliest events in reproduction of humans and mammals that determines whether pregnancy will develop successfully or not. Successful implantation depends on invasive embryo, acceptive endometrium and their synchronization. There is an extremely short period in uterine receptivity for embryo implantation, which is called“implantation window”. Implantation in all mammals involves shedding of zona pellucida, followed by orientation, attachment and adhesion, and invasion of the embryo to the endometrium. Blastocyst implantation is involved in the regulation of large number of genes and the enrollment of complicated regulatory systems.Tumour necrosis factor alpha converting enzyme (TACE) was identified by its ability to cleave tumor necrosis factor alpha at its physiological processing site[1]. It has common sequences of ADAMs: signal peptide, propeptide, catalytic domin, disintegrin domin, cysteine-rich domin, transmembrane domin, cytoplasmic domin[2,3]. By proteolytic removal of the extracellular domain of numerous transmembrane proteins, TACE can be concerned with cell adhesion, fusion, signal transduction and so on. Recent research indicated that TACE was overexpressed in most mammary tumors ,renal cell carcinomas, hepatic cellular cancers, prostatic carcinomas pancreatic carcinomas and colon carcinomas[4,5]. Now it is accepted that the process of embryo implantation is extremely similar with that of tumor invasion and metastasis. Our preliminary studies have shown that the expression of TACE in mouse endometrium during estrus cycle and early pregnancy had regular pattern.The expression in pregnant groups was higher than that in nonpregnant group. TACE expression appeared an increased trend as time passing by, and reached the maximum at pregnancy d 4,TACE expressed mainly in mouse uterine epithelial cell[6].So it is inferred that TACE may play the same role in the process of embryo implantation.Objective:To detect expression of tumor necrosis factor-converting enzyme(TACE) on the mouse uterine epithelial cell and the effect of implantation in vitro. TO analyze its role in blastocyst implantation and to investigate the relation TACE with implantation of mouse embryo.Methods:1.Co-culturing epithelial cells and blastocyst of the pregnant 4-day( d4) mouse in vitro to establish the simple and easy implantation model. 2. Endometrial epithelial cells was identified by Immunochemistry in the co-culture system .3.RT-PCR and Immunochemistry were used to detect the expression of TACE gene and protein in the system.4.Different concentrations of TACE antibody were added to the implantation model to observe effect of TACE antibody on mouse blastocyst implantation in vitro. Expression of TACE protein was detected by Immunochemistry in each antibody interference system.Results:1. Endometrial epithelial cells growed vigorously in vitro.. Keratin antibody showed positive expression, meanwhile, Vimentin antibody showed negative expression in the endometrial epithelial cells.2.PCR showed that the expression of TACE gene was high and results of Immunochemistry , Western-blot were consisted with PCR.3.The adhesion rate of mouse blastocyst was high in the co-culture system and decreased remarkably when TACE antibody was added (P<0.05). The adhesion rate of mouse blastocyst appeared a increased trend as the concentration of TACE antibody lowed. Immunochemistry also showed that expression of TACE protein rised as the concentration of TACE antibody lowed.Conclusion:TACE mRNA and protein expression in mouse endometrium epithelial cells of pregnancy d4 were both strongly high. The abscission of material covered endometrial cells induced by TACE might participate in the process which endometrium changes to the receptive one to accommodate blastocyst implantation. The adhesion rate of mouse blastocyst decreased remarkably when TACE antibody was added . We presumed that TACE participated in the process of mouse blastocyst implantation, which promoted adhesion and outgrowth of blastocyst to prepare for blastocyst implantation.更多还原