本文選題：胚胎著床　切入點(diǎn)：子宮內(nèi)膜　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：胚胎能否成功著床取決于子宮內(nèi)膜和胚泡間的同步協(xié)調(diào)反應(yīng)。在此過程中，子宮內(nèi)膜生理變化所依賴的一系列細(xì)胞因子的差異表達(dá)起著關(guān)鍵作用。有研究表明MicroRNAs （miRNAs）在基因差異表達(dá)的精細(xì)調(diào)控中發(fā)揮了重要作用。課題組前期應(yīng)用miRNAs芯片技術(shù)對(duì)早孕小鼠胚胎著床前后子宮內(nèi)膜miRNAs的表達(dá)進(jìn)行研究，發(fā)現(xiàn)孕6天（d6）和孕4天（d4）相比mmu-miR-141明顯下調(diào)。本文以mmu-miR-141為研究對(duì)象深入探討mmu-miR-141在早孕小鼠胚胎著床前、后子宮內(nèi)膜中的表達(dá)及作用，，為進(jìn)一步了解胚胎著床的分子機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法 1、熒光定量PCR（FQ-PCR）驗(yàn)證芯片結(jié)果，原位雜交方法檢測(cè)mmu-miR-141在早孕小鼠胚胎著床前、后子宮內(nèi)膜組織中的定位表達(dá)； 2、MTT法檢測(cè)mmu-miR-141抑制劑對(duì)小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖的影響，流式細(xì)胞術(shù)檢測(cè)mmu-miR-141抑制劑對(duì)小鼠子宮內(nèi)膜基質(zhì)細(xì)胞周期和細(xì)胞凋亡的影響； 3、應(yīng)用miRNAs的靶基因預(yù)測(cè)數(shù)據(jù)庫檢索mmi-miR-141調(diào)控的靶基因； 4、在子宮內(nèi)膜基質(zhì)細(xì)胞中，分別瞬時(shí)轉(zhuǎn)染mmu-miR-141的模擬物和抑制劑，并用Western Blot檢測(cè)PTEN的表達(dá)； 5、于孕d2天行小鼠子宮角注射mmu-miR-141的模擬物和抑制劑，孕d7天觀察并計(jì)數(shù)胚胎數(shù)。 實(shí)驗(yàn)結(jié)果 1、FQ-PCR結(jié)果顯示mmu-miR-141在著床后（d6）小鼠子宮內(nèi)膜組織中的表達(dá)較著床前（d4）下調(diào)（P0.05），且與芯片結(jié)果一致； 2、原位雜交結(jié)果顯示mmu-miR-141主要定位于基質(zhì)細(xì)胞，在腔上皮和腺上皮中幾乎沒有表達(dá)。著床后（d6）表達(dá)強(qiáng)度較著床前（d4）降低（P0.05）； 3、瞬時(shí)轉(zhuǎn)染mmu-miR-141的抑制劑作用于小鼠子宮內(nèi)膜基質(zhì)細(xì)胞后，MTT檢測(cè)結(jié)果顯示下調(diào)mmu-miR-141的表達(dá)，小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖活性隨之下降（P0.05）。流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示下調(diào)mmu-miR-141表達(dá)時(shí)，細(xì)胞被阻滯在S期，且細(xì)胞凋亡率明顯增加（P0.01）； 5、經(jīng)生物信息學(xué)分析和綜合前期的實(shí)驗(yàn)結(jié)果分析得到mmu-miR-141作用的靶基因有PTEN、PDCD4和KLF6，且PTEN可能性大； 4、采用mmu-miR-141的模擬物或抑制劑分別上調(diào)或下調(diào)mmu-miR-141表達(dá)時(shí)，PTEN的mRNA表達(dá)水平均不受影響。但是，當(dāng)下調(diào)mmu-miR-141的表達(dá)時(shí)，PTEN蛋白表達(dá)顯著增強(qiáng)，而上調(diào)mmu-miR-141的表達(dá)時(shí)，PTEN蛋白表達(dá)水平明顯降低； 6、采用子宮角注射mmu-miR-141的抑制劑和模擬物，均導(dǎo)致胚胎著床數(shù)明顯下降。 結(jié)論 1、mmu-miR-141在早孕小鼠胚胎著床前（d4）后（d6）子宮內(nèi)膜組織中存在著明顯差異的表達(dá)，胚胎著床后較胚胎著床前顯著降低。 2、mmu-miR-141可能通過靶向作用PTEN基因的表達(dá)來調(diào)控子宮內(nèi)膜基質(zhì)細(xì)胞的增殖和凋亡，從而影響小鼠胚胎的著床。
[Abstract]:The successful implantation of an embryo depends on the synchronous and coordinated response between the endometrium and the blastocyst. The differential expression of a series of cytokines dependent on the physiological changes of endometrium plays a key role. Some studies have shown that MicroRNAs miRNAsplays an important role in the fine regulation of gene differential expression. MiRNAs microarray technique was used in the early stage of the study. The expression of miRNAs in endometrium of early pregnant mice before and after implantation was studied. It was found that the expression and role of mmu-miR-141 in the endometrium before and after embryo implantation in early pregnant mice were significantly down-regulated by mmu-miR-141 compared with that on day 6 and day 4 of gestation. It provides experimental basis for further understanding the molecular mechanism of embryo implantation. Method. 1. Fluorescence quantitative PCR FQ-PCRR was used to verify the results of microarray, and in situ hybridization was used to detect the localization of mmu-miR-141 in the endometrium of early pregnant mice before and after implantation. 2the effect of mmu-miR-141 inhibitor on the proliferation of mouse endometrial stromal cells was detected by MTT assay, and the effect of mmu-miR-141 inhibitor on the cell cycle and apoptosis of mouse endometrial stromal cells was detected by flow cytometry. 3. Target genes regulated by mmi-miR-141 were searched by target gene prediction database of miRNAs. (4) in endometrial stromal cells, the mimics and inhibitors of mmu-miR-141 were transiently transfected, and the expression of PTEN was detected by Western Blot. 5. On the 2nd day of gestation, mice were injected with mmu-miR-141 mimics and inhibitors, and the number of embryos was observed and counted on the 7th day of gestation. Experimental results. 1the results of FQ-PCR showed that the expression of mmu-miR-141 in endometrial tissue of postimplantation mice was lower than that of preimplantation day 4 (P 0.05), which was consistent with the results of microarray. 2. The results of in situ hybridization showed that mmu-miR-141 was mainly localized in stromal cells, but hardly expressed in luminal epithelium and glandular epithelium. 3After the transient transfection of mmu-miR-141 inhibitor on mouse endometrial stromal cells, the expression of mmu-miR-141 was down-regulated, and the proliferative activity of mouse endometrial stromal cells decreased (P0.05N). Flow cytometry showed that the expression of mmu-miR-141 was down-regulated. The cells were blocked in S phase and the apoptosis rate was increased significantly (P 0.01). 5. Through bioinformatics analysis and comprehensive analysis of experimental results, we found that the target genes of mmu-miR-141 were PTEN PDCD4 and KLF6, and the possibility of PTEN was high. (4) the expression level of mRNA was not affected when mmu-miR-141 was up-regulated or down-regulated by mmu-miR-141 mimics or inhibitors respectively. However, the expression of mmu-miR-141 protein increased significantly when the expression of mmu-miR-141 was down-regulated, but the expression of mmu-miR-141 protein decreased significantly when the expression of mmu-miR-141 was upregulated. 6. Injection of mmu-miR-141 inhibitor and analogue into uterine horn resulted in a significant decrease in the number of embryo implantation. Conclusion. 1the expression of mmu-miR-141 in endometrium of early pregnant mice was significantly lower than that before implantation, and the expression of mmu-miR-141 in endometrium was significantly lower than that before implantation. 2mmu-miR-141 may regulate the proliferation and apoptosis of endometrial stromal cells by targeting the expression of PTEN gene, thus affecting the implantation of mouse embryos.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 孟君;周生建;魏莎莉;楊戎;覃致遠(yuǎn);馬海蘭;張譚;;PCNA在早孕小鼠子宮內(nèi)膜的表達(dá)[J];生殖與避孕;2008年04期

2 姚楠;陸彩玲;李丹;王沖;孫大光;吳爾若;馬旭;;MicroRNAs在新生鼠卵巢中的表達(dá)研究[J];生殖與避孕;2009年05期

3 黃靜;何俊琳;劉學(xué)慶;丁裕斌;陳世知;陳學(xué)梅;王應(yīng)雄;;β-TrCP和β-連環(huán)蛋白在小鼠胚胎著床過程中子宮內(nèi)膜的表達(dá)[J];細(xì)胞生物學(xué)雜志;2009年02期

4 任剛;楊增明;;MicroRNA在雌性哺乳動(dòng)物生殖中的作用[J];細(xì)胞生物學(xué)雜志;2009年06期

本文編號(hào)：1631350