本文關(guān)鍵詞：小鼠子宮內(nèi)膜mRNAs和miRNAs時(shí)空表達(dá)與胚胎著床的關(guān)系及SPOP對(duì)基質(zhì)細(xì)胞蛻膜化的影響　出處：《重慶醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 微小RNA 深度測(cè)序技術(shù) 胚胎著床 生物信息學(xué) SPOP基因 蛻膜化 增殖 分化

【摘要】：第一部分小鼠子宮內(nèi)膜mRNAs和miRNAs的時(shí)空表達(dá)與胚胎著床的相關(guān)性研究目的：胚胎圍著床是一個(gè)動(dòng)態(tài)的生理過(guò)程,這一過(guò)程被分成三個(gè)階段,分別是容受前期、容受期和胚胎著床期。整個(gè)胚胎圍著床過(guò)程子宮內(nèi)膜形態(tài)和分子發(fā)生顯著改變。子宮內(nèi)膜在不同時(shí)間,不同部位基因差異表達(dá)是引起細(xì)胞組織形態(tài)和分子變化的原因,同時(shí)這也確保了胚胎成功的著床。microRNA(miRNA)是重要的調(diào)節(jié)分子,miRNA主要在細(xì)胞、組織的增殖、分化、凋亡等生理過(guò)程中發(fā)揮作用。而在胚胎著床過(guò)程中miRNA同樣發(fā)揮重要的調(diào)節(jié)作用,miRNAs的對(duì)基因的調(diào)節(jié)作用決定了基因差異表達(dá)的形成。動(dòng)態(tài)分析胚胎著床過(guò)程中mRNA譜和microRNA譜的變化特征,可以為全面理解和闡釋胚胎動(dòng)態(tài)著床過(guò)程中的機(jī)制提供堅(jiān)實(shí)的實(shí)驗(yàn)支持。方法：分別收集容受前期、容受期和胚胎著床期子宮內(nèi)膜組織,對(duì)應(yīng)于小鼠孕第一天(d1)、孕第四天(d4)和孕第五天著床點(diǎn)(d5IS)和孕第五天著床旁(d5IIS)的昆明小鼠子宮內(nèi)膜組織。采用Trizol法提取樣本總RNA。采用深度測(cè)序技術(shù)檢測(cè)小鼠子宮內(nèi)膜不同時(shí)期(d1、d4和d5)或不同部位(d5IS和d5IIS)的mRNA和microRNA的表達(dá)情況。采用RT-PCR檢測(cè)隨機(jī)選擇的miRNA和mR NA表達(dá)水平是否與NGS檢測(cè)的結(jié)果一致。通過(guò)NGS測(cè)序獲得小鼠孕d1、d4、d5IS和d5IIS的mRNA表達(dá)譜,并將四組數(shù)據(jù)進(jìn)行兩兩成對(duì)比較,篩選出顯著性差異表達(dá)基因,采用維恩圖對(duì)小鼠圍著床期基因表達(dá)進(jìn)行動(dòng)態(tài)分析。通過(guò)NGS測(cè)序獲得小鼠d1、d4、d5IS和d5IIS的miRNA表達(dá)譜,通過(guò)兩兩成對(duì)比較獲得顯著性差異的miRNAs,再利用Targetscan database、 miRanda database和DIANA-microT 3.0三種軟件預(yù)測(cè)miRNA的靶向基因,最后預(yù)測(cè)結(jié)果采取交集定為最終的靶基因。利用mRNA譜確定靶基因的表達(dá)量,同時(shí)結(jié)合miRNA表達(dá)量篩選出miRNA與對(duì)應(yīng)靶向基因是否具有負(fù)向調(diào)節(jié)的關(guān)系。結(jié)果：NGS檢測(cè)的miRNAs結(jié)果表明d 1、d4、d5IS和d5IIS的miRNAs表達(dá)具有顯著差異,其中d1和d5IS組織中以上調(diào)miRNAs表達(dá)為主；而d4組織中以下調(diào)的miRNAs表達(dá)為主。提示在圍著床期miRNAs具有不同的作用。對(duì)顯著差異表達(dá)的63個(gè)miRNAs進(jìn)行靶基因預(yù)測(cè),獲得5720個(gè)靶基因。結(jié)合miRNAs譜和mRNAs譜的表達(dá)水平,在d1、d4、d5三個(gè)時(shí)間點(diǎn)共篩選出1473對(duì)miRNA-mRNA呈現(xiàn)負(fù)相關(guān)性表達(dá),而在d1、d4、d5三個(gè)時(shí)間點(diǎn)miRNA-mRNA表達(dá)量都始終保持負(fù)相關(guān)的有138對(duì)。在負(fù)相關(guān)表達(dá)的miRNA-mRNA對(duì)中隨機(jī)選取5對(duì)miRNA-mRNAs利用實(shí)時(shí)熒光定量PCR進(jìn)行檢測(cè)miRNA-mRNAs各自的表達(dá)量,驗(yàn)證了NGS檢測(cè)的miRNA和mRNA數(shù)據(jù)譜可靠性,同時(shí)證明了在圍著床期miRNA-mRNA存在負(fù)相關(guān)性,表明miRNA對(duì)mRNA具有調(diào)控作用。根據(jù)miRNA-mRNA相互作用網(wǎng)絡(luò)圖分析,表明了miRNAs對(duì)mRNAs的調(diào)節(jié)作用。同時(shí)揭示了與容受態(tài)功能相關(guān)的Bcl-2,Klf13和PGR基因是網(wǎng)絡(luò)圖中核心miRNA:mmu-miR-106a-5p,mmu-miR-200b-3p,mmu-miR-96-5p的靶基因。結(jié)論：NGS檢測(cè)的mRNAs結(jié)果表明孕d1、d4、d5IS和d5IIS的mRNA表達(dá)具有顯著時(shí)空差異性,這種表達(dá)差異決定了圍著床期子宮內(nèi)膜在不同時(shí)期和部位發(fā)揮不同作用。在胚胎著床時(shí)miRNAs對(duì)mRNAs表達(dá)存在調(diào)控作用。而這種調(diào)節(jié)方式在圍著床期的整個(gè)過(guò)程持續(xù)發(fā)揮作用。多個(gè)與與容受態(tài)相關(guān)的Bcl-2,Klf13和PGR基因是核心miRNA的靶基因表明miRNAs在小鼠子宮內(nèi)膜容受狀態(tài)的形成和維持早期妊娠中發(fā)揮重要作用。第二部分SPOP對(duì)子宮內(nèi)膜基質(zhì)細(xì)胞蛻膜化的影響目的：子宮內(nèi)膜基質(zhì)細(xì)胞蛻膜分化是成功實(shí)現(xiàn)胚胎著床的重要過(guò)程,受雌孕激素調(diào)節(jié)的子宮內(nèi)膜基質(zhì)細(xì)胞在這一過(guò)程發(fā)生明顯的增殖、分化。我們前期研究證明,SPOP在胚胎著床第5天的著床點(diǎn)和著床旁組織中表達(dá)存在顯著差異,而SPOP與泛素化作用密切相關(guān)。關(guān)于泛素化與胚胎著床的相關(guān)研究尚未見(jiàn)報(bào)道。因此,本課題著眼于小鼠圍著床期SPOP研究,揭示SPOP在胚胎著床過(guò)程中發(fā)揮的作用。方法：建立昆明小鼠早孕模型、小鼠假孕模型、人工激素調(diào)控的小鼠模型。采用定量PT-PCR、Western blot和免疫組織化學(xué)技術(shù)檢測(cè)SPOP mRNA或蛋白在各種模型小鼠(早孕小鼠,假孕小鼠和激素調(diào)控)子宮內(nèi)膜中表達(dá)規(guī)律。采用siRNA構(gòu)建人工誘導(dǎo)蛻膜細(xì)胞SPOP敲除模型。siRNA敲除SPOP后,檢測(cè)蛻膜化細(xì)胞蛻膜標(biāo)志物的表達(dá)情況。利用流式細(xì)胞技術(shù)檢測(cè)SPOP對(duì)凋亡作用的影響。結(jié)果：SPOP在圍著床期呈波動(dòng)表達(dá)dl、d2和d3表達(dá)較低,從d4到d6表達(dá)顯著增加,d7表達(dá)出現(xiàn)回落。假孕小鼠模型中SPOP具有同樣波動(dòng)表達(dá)的模式。人工誘導(dǎo)蛻膜化后小鼠子宮內(nèi)膜SPOP表達(dá)顯著增加。利用激素調(diào)控小鼠模型證實(shí)SPOP的表達(dá)受雌激素和孕激素的調(diào)控。siRNA敲除SPOP后基質(zhì)蛻膜細(xì)胞的蛻膜標(biāo)志物(IGFBP-1和C yclinD3)表達(dá)水平顯著降低。SPOP能夠抑制基質(zhì)細(xì)胞凋亡發(fā)生結(jié)論：雌激素、孕激素能夠調(diào)節(jié)SPOP的表達(dá),SPOP通過(guò)抑制凋亡、泛素化作用影響基質(zhì)細(xì)胞蛻膜化發(fā)生,能促進(jìn)基質(zhì)細(xì)胞蛻膜分化。
[Abstract]:The first part of the study of space-time expression of mRNAs and miRNAs between mouse endometrium and embryo implantation Objective: embryo implantation is physiological around a dynamic process, this process is divided into three stages, namely early stage and suffer, suffer during embryo implantation. The whole embryo around the bed of endometrial morphology and molecular change. The endometrium at different time and different position of gene expression is the cause of the morphological and molecular changes of cells and tissues, it also ensures the successful embryo implantation of.MicroRNA (miRNA) is an important regulatory molecule, mainly in miRNA cells, proliferation, differentiation, apoptosis and other physiological processes play a role in embryo. In the process of implantation miRNA also play an important role in the regulation of miRNAs gene, to determine the form of differential gene expression. Dynamic analysis of embryo implantation process mR NA spectrum and microRNA spectrum characteristics, can provide solid support for the comprehensive understanding and interpretation of the dynamic mechanism in the process of embryo implantation. Methods: during the early stage and tissue in endometrium during embryo implantation, corresponding to the first day of pregnant mice (D1), day fourth of gestation (D4) and at Fifth implantation sites (d5IS) and at day fifth (d5IIS) of Kunming by implantation of mouse endometrial tissue. The sample was extracted by Trizol total RNA. using deep sequencing technology to detect mouse endometrium in different periods (D1, D4 and D5) or different parts (d5IS and d5IIS) expression of mRNA and microRNA by RT-PCR. To detect the expression of miRNA and mR NA were randomly selected and NGS level detection results. Get pregnant D1 mice by NGS sequencing, D4, d5IS and d5IIS mRNA expression, and the data of the four groups were screened out 22 pairwise comparison, significant difference table As the gene, a Venn diagram for dynamic analysis of periimplantation. Gene expression of mouse D1 by NGS sequencing, D4, d5IS and d5IIS miRNA expression by 22, obtained significant difference pairwise comparison of miRNAs, then Targetscan database, miRanda database and DIANA-microT 3 three miRNA target gene prediction software finally, the prediction results taken as the final intersection of target gene. Target gene expression spectrum determined by mRNA, combined with the expression of miRNA miRNA was screened with the corresponding target gene has negative regulation relationship. Results: the detection of NGS miRNAs results showed that D 1, D4, d5IS and d5IIS miRNAs expression significant differences among D1 and d5IS tissues above miRNAs expression; D4 and tissue miRNAs expression. The following tips have different effects during the peri implantation period miRNAs. Significant differences on table 63 miRNAs as the target gene prediction, 5720 target genes. Combined with the expression of miRNAs and mRNAs spectra in D1, D4, D5 three time points were selected from 1473 of miRNA-mRNA showed a negative correlation expression, while in the D1, D4, D5 expression in three time points of miRNA-mRNA were maintained the negative correlation of 138. In the negative expression of miRNA-mRNA on 5 randomly selected for miRNA-mRNAs using real-time fluorescence quantitative PCR to detect the expression of miRNA-mRNAs of each volume, verify the detection of NGS miRNA and mRNA spectral data reliability, and that during the peri implantation period there is a negative correlation between the miRNA-mRNA, show that miRNA has a role in the regulation of mRNA according to the analysis of miRNA-mRNA interaction network, shows that the effect of miRNAs on the mRNAs. At the same time reveals the related and receptivity state function of Bcl-2, Klf13 and PGR genes in the network diagram of core miRNA: mmu-miR-106a-5p, mmu-miR- 200b-3p, the target gene of mmu-miR-96-5p. Conclusion: NGS mRNAs results showed that D4, d5IS. D1, d5IIS and mRNA expression have significant temporal differences, the expression difference of periimplantation endometrium play different roles in different stages and parts. The expression of miRNAs in embryo implantation has regulatory effect on mRNAs. This adjustment in the periimplantation period the whole process continued to play a role. Multiple and receptivity state related Bcl-2, Klf13 and PGR gene is the target gene core miRNA show that miRNAs in mice endometrial receptivity in the formation and maintenance of early pregnancy play an important role. The second part SPOP of endometrial stromal cell decidualization Objective: endometrial stromal cells in decidual differentiation is the successful implementation of an important process of embryo implantation, the occurrence in the process of estrogen and progesterone regulation of endometrial stromal cells. Obvious proliferation and differentiation. Our previous studies have proved that there are significant differences in the expression of SPOP in embryo implantation fifth days of implantation site and adjacent tissues, while SPOP and ubiquitination are closely related. Related research about the ubiquitination and embryo implantation has not been reported. Therefore, this study focused on mice of SPOP bed around the stage reveal, SPOP play in embryo implantation process. Methods: Kunming mice model of early pregnancy, pseudopregnancy mice model, mice model of artificial hormone regulation. By quantitative PT-PCR, Western blot and immunohistochemical detection of SPOP or mRNA proteins in various model mice (mouse, pseudopregnant mice and hormonal regulation of endometrial expression) in order to construct knockout model..siRNA knockdown of SPOP after artificial induction of SPOP in decidual cells by siRNA, detection of decidualization marker decidual cells expression by flow cytometry. Effect of detection of SPOP on apoptosis. Results: SPOP during the peri implantation period fluctuated expression of DL, D2 and D3 had low expression, from D4 to D6 was significantly increased, the expression of D7 declined. SPOP pseudopregnant mice model with the same expression pattern. Fluctuations induced by small pieces of endometrial SPOP expression increased significantly in decidua after using the mouse model confirmed the hormone regulation. The regulation of.SiRNA SPOP expression by estrogen and progesterone knockout SPOP stromal decidual cell decidual markers (IGFBP-1 and C yclinD3) expression level was significantly lower in.SPOP can inhibit the apoptosis of stromal cells. Conclusion: estrogen, progesterone can regulate the expression of SPOP and SPOP by inhibiting apoptosis, ubiquitin the occurrence of stromal cell decidualization of stromal cells can promote decidual differentiation.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R321.3

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3 成鷹;大腸桿菌和布魯氏菌脂多糖刺激條件下巨噬細(xì)胞差異表達(dá)miRNAs的鑒定及其作用機(jī)制[D];海南大學(xué);2014年

4 王奕;MiRNAs在白癜風(fēng)外周血單個(gè)核細(xì)胞中的表達(dá)及miR-3940-5p對(duì)T細(xì)胞作用機(jī)制的研究[D];山東大學(xué);2015年

5 陳科;小鼠子宮內(nèi)膜mRNAs和miRNAs時(shí)空表達(dá)與胚胎著床的關(guān)系及SPOP對(duì)基質(zhì)細(xì)胞蛻膜化的影響[D];重慶醫(yī)科大學(xué);2015年

6 于鵬;miRNAs對(duì)骨髓間充質(zhì)干細(xì)胞分化的影響[D];北京協(xié)和醫(yī)學(xué)院;2013年

7 吳紹華;不同倍性水稻miRNAs及其靶基因的鑒定與表達(dá)分析[D];四川農(nóng)業(yè)大學(xué);2011年

8 陳毅鵬;酒精性肝病特異性miRNAs表達(dá)譜及下游調(diào)控基因研究[D];浙江大學(xué);2014年

9 牛文洋;肝細(xì)胞癌血清特異miRNAs的篩選及其在診斷中的應(yīng)用[D];第二軍醫(yī)大學(xué);2011年

10 郭玲;UVB相關(guān)的miRNAs的篩選及其功能的初步研究[D];南方醫(yī)科大學(xué);2010年

相關(guān)碩士學(xué)位論文 前10條

1 姜青華;丹參miRNAs組織特異表達(dá)譜及其靶基因鑒定[D];杭州師范大學(xué);2015年

2 劉元會(huì);抑郁患者腦脊液和血清中差異性miRNAs鑒定的臨床研究[D];四川醫(yī)科大學(xué);2015年

3 賈文慧;宮頸癌及子宮內(nèi)膜癌血清miRNAs的研究[D];南京大學(xué);2013年

4 許成辰;不同HER-2水平的人乳腺癌MCF-7細(xì)胞內(nèi)miRNAs表達(dá)譜及相關(guān)miRNAs對(duì)癌細(xì)胞生物學(xué)行為的影響[D];安徽醫(yī)科大學(xué);2015年

5 曹文婷;奶山羊乳腺組織miRNAs的鑒定、篩選及功能的初步研究[D];西北農(nóng)林科技大學(xué);2015年

6 劉帥帥;循環(huán)miRNAs檢測(cè)對(duì)肝癌診斷價(jià)值的meta分析[D];蚌埠醫(yī)學(xué)院;2015年

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8 雷彬;大白豬和二花臉豬排卵前卵泡差異表達(dá)miRNAs及其靶基因鑒定[D];華中農(nóng)業(yè)大學(xué);2013年

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10 孔德艷;水稻抗旱相關(guān)miRNAs的克隆及其功能的初步研究[D];華中農(nóng)業(yè)大學(xué);2010年

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