本文關(guān)鍵詞：骨橋蛋白通過MAPK信號通路上調(diào)MMP9表達(dá)及與胚胎著床相關(guān)性分析　出處：《大連醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨橋蛋白(OPN) 基質(zhì)金屬蛋白酶 9(MMP9) MAPK 信號通路 RL95-2 細(xì)胞 胚胎著床

【摘要】：目的:研究骨橋蛋白(OPN)在圍著床窗口期的小鼠子宮內(nèi)膜的變化規(guī)律,探討在人子宮內(nèi)膜腫瘤細(xì)胞RL95-2細(xì)胞(用來模擬著床期子宮內(nèi)膜細(xì)胞)中,OPN能否通過某一信號通路促進(jìn)MMP9的表達(dá),分析在胚胎著床的過程中OPN所發(fā)揮的作用及機制。 方法:(1)提取妊娠第1、2、3、4、5天(D1- D5)的小鼠的子宮內(nèi)膜上皮組織蛋白和mRNA,用Western blot方法和RT-PCR方法檢測OPN的表達(dá)的變化情況(。2)用重組的OPN蛋白加入到無血清無雙抗條件的培養(yǎng)液中刺激RL95-2細(xì)胞,用ELISA方法檢測RL95-2細(xì)胞MMP9表達(dá)的變化情況和相應(yīng)通路的變化情況。(3)構(gòu)建攜帶有氨芐抗性表達(dá)基因和OPN變異體A(OPN全序列)的質(zhì)粒,轉(zhuǎn)染入RL95-2細(xì)胞中,檢測轉(zhuǎn)染后培液中和RL95-2細(xì)胞中MMP9的蛋白和mRNA表達(dá)情況,分析相應(yīng)通路的變化情況 結(jié)果:(1)妊娠期間小鼠子宮內(nèi)膜OPN的表達(dá)變化明顯,在妊娠D1-4天隨著妊娠天數(shù)的增加而增加,在D4天小鼠胚胎著床窗口期表達(dá)達(dá)到最高峰,D5天表達(dá)量開始下降。(2)用OPN重組蛋白刺激RL95-2細(xì)胞后,MMP9的表達(dá)量明顯增加,并與OPN重組蛋白的加入量有著劑量依賴性,即隨著OPN重組蛋白加入量的增加而增加,并且OPN能夠通過ERK1/2的磷酸化而激活MAPK信號通路促使下游分子MMP9的表達(dá)上調(diào),且OPN刺激ERK1/2的磷酸化隨著時間的增加而增加,在刺激1小時后達(dá)到最高值,但在2h時既開始下降。(3)OPN質(zhì)粒構(gòu)建成功,RL95-2細(xì)胞的轉(zhuǎn)染效率低下,轉(zhuǎn)染方法有待改進(jìn)。 結(jié)論:(1)OPN在妊娠開始階段到胚胎著床階段的表達(dá)呈遞增趨勢,并在著床窗口期呈現(xiàn)峰值,著床后其表達(dá)有所下降,所以O(shè)PN很有可能參與胚胎著床的過程。(2)OPN參與胚胎著床的機制之一可能是通過磷酸化ERK1/2激活MAPK信號通路促進(jìn)下游分子基質(zhì)金屬蛋白酶9 MMP9的表達(dá),水解子宮內(nèi)膜上皮細(xì)胞以利于胚胎著床。(3)成功構(gòu)建OPN質(zhì)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R321

【參考文獻(xiàn)】

中國期刊全文數(shù)據(jù)庫 前2條

1 江平;謝青貞;;骨橋蛋白與胚胎著床及胚胎發(fā)育的研究進(jìn)展[J];國際生殖健康/計劃生育雜志;2009年04期

2 孫曉玲;衛(wèi)慶萍;于樹芳;豆瓊;范路霞;;骨橋蛋白(OPN)在婦科與生殖領(lǐng)域中的研究進(jìn)展[J];中國優(yōu)生與遺傳雜志;2010年10期

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本文編號：1413133