骨形态发生蛋白-7（BMP-7）在小鼠蜕膜细胞的表达及功能研究

发布时间：2018-06-23 21:39

本文选题：骨形态发生蛋白-7(BMP-7) + 子宫内膜　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的:胚胎着床是胎生哺乳动物及人类生殖生理的重要环节,是母体子宫与胚胎同步发育、在特定时期即“着床窗”开放期间精确调节、快速完成的生理现象。妊娠需要子宫组织发生广泛的重建,子宫内膜细胞生长和分化,即发生蜕膜化。子宫内膜的蜕膜化过程对于成功的着床和妊娠的维持是必需的。蜕膜化过程涉及黏附分子、蛋白水解酶、生长因子、细胞因子、血管活性因子以及多种基因的相互作用和调节。已有研究表明BMPs家族在小鼠胚胎发育过程中都有表达,为进一步研究其家族中BMP-7在小鼠子宫中的表达规律和作用,本实验采用细胞原代培养、荧光定量PCR、免疫组织化学、免疫细胞化学、Western blotting等方法从mRNA和蛋白水平检测BMP-7在妊娠小鼠子宫中的表达,探索其在子宫内膜蜕膜化过程中的作用。方法: 1.RT-PCR和免疫组织化学技术分别观察BMP-7的mRNA和蛋白在妊娠第5-8日小鼠子宫中的表达情况;2.原代培养小鼠子宫基质细胞,用孕酮、雌二醇和cAMP诱导蜕膜化,并在诱导蜕膜化的同时分别加入1.0ng/ml、10ng/ml、100ng/ml BMP-7重组腺病毒,于24h、48h、72h、96h检测催乳素的表达;3.去卵巢小鼠随机分3组,分别皮下注射芝麻油(0.1ml/只)、孕酮(2.0mg/只)、RU486(2.0mg/只)+孕酮(2.0mg/只),于注射后0h、6h、12h、24h处死小鼠收集子宫,提取子宫RNA和总蛋白,荧光定量PCR、Western blotting检测BMP-7的表达情况。结果:1.妊娠第5-8日,BMP-7 mRNA在小鼠子宫着床位点的表达高于着床旁组织(P0.01),且在着床点的表达随着妊娠天数的增加逐渐增强(P0.05)。妊娠第5日、第6日,BMP-7蛋白分别表达于植入胚胎周围的基质细胞和初级蜕膜带;第7日、第8日,主要表达于次级蜕膜带以及植入位点系膜侧基质细胞。2.在小鼠子宫基质细胞的蜕膜化过程中,BMP-7重组腺病毒组催乳素的mRNA和蛋白表达均显著增加(P0.01),其中10ng/ml组增加最明显(P0.01)。3.去卵巢小鼠模型研究显示:孕酮上调了小鼠子宫组织BMP-7的表达(P0.01),孕酮受体拮抗剂RU486能有效阻断孕酮对BMP-7的上调作用(P0.05) 结论:BMP-7基因在小鼠子宫内的表达受孕酮调控,具有促进小鼠子宫内膜蜕膜化的效应。
[Abstract]:Objective: embryo implantation is an important link in reproductive physiology of fetal mammals and human beings. It is a physiological phenomenon that the womb and embryo develop synchronously and regulate accurately and finish quickly during the opening period of implantation window. Pregnancy requires extensive reconstruction of uterine tissue, endometrial cell growth and differentiation, that is, decidualization. Decidualization of the endometrium is essential for successful implantation and pregnancy maintenance. Decidualization involves the interaction and regulation of adhesion molecules, proteolytic enzymes, growth factors, cytokines, vasoactive factors and multiple genes. In order to further study the expression of BMP-7 in mouse uterus, the primary cell culture, fluorescence quantitative PCR and immunohistochemistry were used to study the expression of BMP-7 in mouse uterus. The expression of BMP-7 in the uterus of pregnant mice was detected by immunocytochemistry and Western blotting, and the role of BMP-7 in decidualization of endometrium was explored. Methods: 1. RT-PCR and immunohistochemistry were used to observe the expression of BMP-7 mRNA and protein in the uterus of mice on the 5th and 8th day of pregnancy respectively. Primary cultured mouse uterine stromal cells were induced decidualization by progesterone, estradiol and camp. The recombinant adenovirus BMP-7 was added with 1.0 ng / ml 10ng / ml BMP-7 respectively. Prolactin expression was detected at 24 h, 48 h, 72 h and 96 h, respectively. Ovariectomized mice were randomly divided into three groups: subcutaneously injected with sesame oil (0.1ml/), progesterone (2.0mg/), RU486 (2.0mg/), progesterone (2.0mg/). The mice were killed at 0 h, 6 h, 12 h and 24 h after injection to collect uterus, extract uterine RNA and total protein, and detect the expression of BMP-7 by Western blotting. The result is 1: 1. The expression of BMP-7 mRNA on the 5th to 8th day of pregnancy was higher than that in the para-implantation tissue (P0.01), and the expression of BMP-7 mRNA increased gradually with the increase of pregnancy days (P0.05). On the 5th and 6th day of pregnancy, BMP-7 protein was expressed in stromal cells and primary decidua band around implantation embryos, and mainly in secondary decidua band and Mesangial side stromal cells at implantation site on the 7th and 8th day of gestation. During decidualization of mouse uterine stromal cells, prolactin mRNA and protein expression in BMP-7 recombinant adenovirus group increased significantly (P0.01), especially in 10ng/ml group (P0.01). The study of ovariectomized mouse model showed that progesterone up-regulated the expression of BMP-7 in mouse uterus (P0.01). RU486, a progesterone receptor antagonist, could effectively block the up-regulation of BMP-7 by progesterone (P0.05). The expression of progesterone is regulated by progesterone. It has the effect of promoting decidualization of mouse endometrium.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R321

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