【作者】 梁晓欢；

【导师】 杨增明；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2009， 博士

【摘要】 胚胎着床需要子宫内膜接受态的建立以及胚胎发育到具有黏附能力的胚泡同步进行。胚胎着床开始后的蜕膜化过程中,子宫内膜基质细胞发生增殖和分化。在胚胎着床和蜕膜化过程中,组胺和多胺等胺类发挥着重要的作用。来源于母体子宫的组胺与胚胎上的组胺受体相互作用有利于着床过程。细胞中多胺水平的平衡对于维持细胞增殖等过程非常重要。但这些具有生物活性的胺类的过量积累,会破坏细胞稳态,甚至导致细胞凋亡。阿米洛利结合蛋白1(amiloride binding protein 1,Abp1)是哺乳动物体内代谢组胺和腐胺等胺类的主要降解酶,对于维持生物胺类水平的平衡具有很重要的作用。本实验利用原位杂交、实时定量PCR和Western blot等方法,检测了Abp1在小鼠早期妊娠子宫中的表达,并利用假孕、延迟着床、人工诱导蜕膜化和激素处理等多种模型研究了Abp1在子宫中的调节。在体外细胞培养水平,我们检测了类固醇激素对Abp1的调节,以及在诱导蜕膜化过程中Abp1表达的变化。此外,通过启动子及荧光素酶分析等方法,我们研究了Cebpb和cAMP在早期妊娠过程中对Abp1的调节作用。Abp1 mRNA在妊娠第5天子宫着床位点处腔上皮下基质中显著上调,之后持续表达于正在发生蜕膜化的基质细胞中,并随着妊娠过程的发展,在蜕膜区的表达逐渐增强。在假孕第1-5天以及延迟着床子宫中检测不到Abp1的表达,而在延迟后激活子宫的着床位点,Abp1的表达增强。人工诱导蜕膜化模型中,对照侧子宫中检测不到Abp1的表达,而诱导蜕膜化的子宫中Abp1大量表达。通过体外诱导蜕膜化模型发现,在诱导蜕膜化的细胞中,Abp1表达增强。我们证实,在Abp1启动子上存在cAMP的反应元件,cAMP处理能够上调Abp1的表达。由于Abp1的表达主要定位于子宫内膜的基质细胞中,将培养的子宫基质细胞用类固醇激素处理时,Abp1的表达受孕酮上调,雌激素对孕酮的上调效应起抑制作用。孕酮受体拮抗剂RU486处理可逆转孕酮对Abp1的上调作用,说明孕酮通过其受体PR实现对Abp1的调节。通过启动子分析,在Abp1的启动子上找到了转录因子CCAAT/增强子结合蛋白β(Cebpb)的结合位点。利用双荧光素酶分析,证实Cebpb对Abp1具有上调作用。当把Abp1启动子上Cebpb的结合位点突变后,Cebpb对Abp1的上调作用显著降低。我们的结果表明,Abp1的表达受活性胚胎的调节,且在蜕膜化过程中表达逐渐增强。Abp1的表达受孕酮、cAMP和Cebpb上调。Abp1可能在小鼠胚胎着床及蜕膜化过程起重要作用。更多还原

【Abstract】 Embryo implantation requires a precise synchronism between the receptive uterus and activated blastocyst. Embryo implantation is followed by decidualization, when stromal cells go through proliferation and differentiation. Histamine and polyamines play key roles in pregnancy. Uterine-derived histamine interacts with embryonic H2 receptors in a paracrine fashion to initiate the process of implantation; the balance of polyamines is important for cell proliferation and differentiation. Amiloride binding protein 1 (Abp1), an amine oxidase involved in the metabolism of histamine and putrescine, is important in the regulation of amine levels. In situ hybridization, real-time PCR and western blot was preformed to detect the expression of Abp1 in mouse uterus during early pregnancy. We also used pseudopregnancy, delayed implantation and activation, artificial decidualization and hormonal treatment models to study the regulation of Abp1 in uterus. In cultured uterine stromal cells, we examined the regulation of steroid hormones and induced decidualization on Abp1 expression. Using promoter assay, the effects of Cebpb and cAMP on Abp1 was also investigated.On day 5 of pregnancy, a high level of Abp1 mRNA signal was found in the subluminal stroma immediately surrounding the implanting blastocyst. From days 6 to 8, Abp1 was highly expressed in the primary decidua and increased during decidualization. There was no detectable Abp1 mRNA signal in the uterus from days 1 to 5 of pseudopregnancy or under delayed implantation. After delayed implantation was terminated by estrogen treatment, Abp1 expression was observed in the subluminal stroma cells surrounding the implanting blastocyst. Under artificial decidualization, no Abp1 mRNA signal was detected in the control horn, but the expression was strongly seen in the decidualized cells. Induction of decidualization in vitro confirmed the up-regulation of Abp1 expression during decidualization. cAMP treatment also elevated the level of Abp1. The endometrial stromal cells cultured in vitro was also used to determine whether steroid hormones could regulate Abp1 expression. In the cultured stromal cells, Abp1 expression was significantly stimulated by progesterone. Estrogen attenuated the up-regulated effect of progesterone. RU486 treatment before progesterone reversed the up-regulation of Abp1 by progesterone. These results showed that Abp1 is a downstream target of PR. With promoter assay, transcriptional factors CCAAT/enhancer binding proteinβ(Cebpb) binding sites were found in the promoter region of mouse Abp1 gene. Dual-luciferase activity assay confirmed the regulation of Cebpb on Abp1 expression. Abp1 promoter activity was significantly induced in primary uterine stromal cells by Cebpb. Site-directed mutagenesis of the Cebpb binding sites in Abp1 promoter abolished the upregulation of Abp1 promoter activity. Our results showed that the expression of Abp1 was dependent on the presence of active blastocyst and increased during the process of decidualization. Abp1 expression was under the regulation of progesterone, cAMP and Cebpb. In conclusion, our data suggested that Abp1 may play a key role during implantation and decidualization.更多还原