骨桥蛋白通过MAPK信号通路上调MMP9表达及与胚胎着床相关性分析

发布时间：2018-01-12 06:34

  本文关键词：骨桥蛋白通过MAPK信号通路上调MMP9表达及与胚胎着床相关性分析　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 骨桥蛋白(OPN) 基质金属蛋白酶 9(MMP9) MAPK 信号通路 RL95-2 细胞 胚胎着床

【摘要】：目的:研究骨桥蛋白(OPN)在围着床窗口期的小鼠子宫内膜的变化规律,探讨在人子宫内膜肿瘤细胞RL95-2细胞(用来模拟着床期子宫内膜细胞)中,OPN能否通过某一信号通路促进MMP9的表达,分析在胚胎着床的过程中OPN所发挥的作用及机制。 方法:(1)提取妊娠第1、2、3、4、5天(D1- D5)的小鼠的子宫内膜上皮组织蛋白和mRNA,用Western blot方法和RT-PCR方法检测OPN的表达的变化情况(。2)用重组的OPN蛋白加入到无血清无双抗条件的培养液中刺激RL95-2细胞,用ELISA方法检测RL95-2细胞MMP9表达的变化情况和相应通路的变化情况。(3)构建携带有氨苄抗性表达基因和OPN变异体A(OPN全序列)的质粒,转染入RL95-2细胞中,检测转染后培液中和RL95-2细胞中MMP9的蛋白和mRNA表达情况,分析相应通路的变化情况 结果:(1)妊娠期间小鼠子宫内膜OPN的表达变化明显,在妊娠D1-4天随着妊娠天数的增加而增加,在D4天小鼠胚胎着床窗口期表达达到最高峰,D5天表达量开始下降。(2)用OPN重组蛋白刺激RL95-2细胞后,MMP9的表达量明显增加,并与OPN重组蛋白的加入量有着剂量依赖性,即随着OPN重组蛋白加入量的增加而增加,并且OPN能够通过ERK1/2的磷酸化而激活MAPK信号通路促使下游分子MMP9的表达上调,且OPN刺激ERK1/2的磷酸化随着时间的增加而增加,在刺激1小时后达到最高值,但在2h时既开始下降。(3)OPN质粒构建成功,RL95-2细胞的转染效率低下,转染方法有待改进。 结论:(1)OPN在妊娠开始阶段到胚胎着床阶段的表达呈递增趋势,并在着床窗口期呈现峰值,着床后其表达有所下降,所以OPN很有可能参与胚胎着床的过程。(2)OPN参与胚胎着床的机制之一可能是通过磷酸化ERK1/2激活MAPK信号通路促进下游分子基质金属蛋白酶9 MMP9的表达,水解子宫内膜上皮细胞以利于胚胎着床。(3)成功构建OPN质粒。
[Abstract]:Objective: to study the changes of osteopontin (OPN) in the endometrium of mice during the period of surrounding bed window. To investigate whether RL95-2 can promote the expression of MMP9 through a signal pathway in human endometrial tumor cells (which is used to mimic the implantation phase of endometrial cells). To analyze the role and mechanism of OPN in embryo implantation. Methods the endometrial epithelium protein and mRNA were extracted from mice with D1-D5 on the first day of gestation. Detection of OPN expression by Western blot and RT-PCR. The recombinant OPN protein was added to the medium without serum-free double antibody to stimulate RL95-2 cells. Using ELISA method to detect the changes of MMP9 expression in RL95-2 cells and the changes of the corresponding Pathway. 3) Construction of Amphixil resistant expression Gene and OPN variant A( 1). The plasmid of OPN. After transfection into RL95-2 cells, the expression of MMP9 protein and mRNA in culture medium and RL95-2 cells after transfection were detected, and the changes of the corresponding pathway were analyzed. Results the expression of OPN in endometrium of mice was significantly changed during pregnancy. The expression of OPN increased with the increase of gestational days on day 1-4 of pregnancy, and reached the peak at window stage of embryo implantation on day D4. The expression of MMP9 in RL95-2 cells was significantly increased after stimulation with OPN recombinant protein, which was in a dose-dependent manner with the addition of OPN recombinant protein. That is, with the increase of the amount of OPN recombinant protein, OPN can activate the MAPK signaling pathway through the phosphorylation of ERK1/2 and promote the up-regulation of downstream MMP9 expression. The phosphorylation of ERK1/2 stimulated by OPN increased with the increase of time, and reached the highest level after 1 hour of stimulation, but it began to decrease at 2 h, and the plasmid was successfully constructed. The transfection efficiency of RL95-2 cells was low and the transfection method needed to be improved. Conclusion the expression of OPN increased from the beginning of pregnancy to the stage of embryo implantation, and showed a peak value at the window of implantation, and the expression of OPN decreased after implantation. So OPN is likely to be involved in embryo implantation. One of the mechanisms of OPN involved in embryo implantation may be that phosphorylation of ERK1/2 activates MAPK signaling pathway to promote the expression of downstream matrix metalloproteinase-9 MMP9. OPN plasmid was successfully constructed by hydrolysis of endometrial epithelial cells to facilitate embryo implantation.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321

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