本文選題：microRNA-214 + QKI　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：研究背景及目的:以動脈粥樣硬化為基本組織病理改變的血管損傷性病變,其基本病理進(jìn)程為:血管損傷引起血管修復(fù)和重塑,繼而血管內(nèi)膜增厚,導(dǎo)致管腔狹窄。在這一病變過程中,血管平滑肌細(xì)胞通過表型轉(zhuǎn)換、增殖、遷移以及干細(xì)胞分化兩種方式參與其中。既往研究已經(jīng)發(fā)現(xiàn)microRNA-214(miR-214)調(diào)控成熟血管平滑肌細(xì)胞功能和表型轉(zhuǎn)換,然而對于其在干細(xì)胞向血管平滑肌細(xì)胞分化中的作用尚不是十分清楚。本研究希望揭示miR-214在胚胎干細(xì)胞向血管平滑肌細(xì)胞分化中的作用,鑒定miR-214的靶基因,闡明其作用機(jī)制。研究方法:本研究主要利用課題組前面建立的、穩(wěn)定的、體外的小鼠胚胎干細(xì)胞向平滑肌細(xì)胞分化模型,觀察miR-214表達(dá)變化,并過表達(dá)或抑制miR-214表達(dá),研究miR-214對胚胎干細(xì)胞向血管平滑肌細(xì)胞分化的作用。其次,利用生物信息學(xué)技術(shù)分析及螢光素酶基因報告系統(tǒng)等多種方法鑒定及確認(rèn)miR-214的功能性靶基因。再者,利用免疫共沉淀(IP)、雙熒光素酶基因報告系統(tǒng)等多種分子實驗技術(shù),證明QKI對胚胎干細(xì)胞向血管平滑肌細(xì)胞分化的作用及作用途徑。最后,利用體外轉(zhuǎn)染的細(xì)胞打入動物體內(nèi)模擬在體環(huán)境的小鼠胚胎干細(xì)胞分化模型,研究miR-214對胚胎干細(xì)胞向平滑肌細(xì)胞分化的影響。結(jié)果:利用課題組以前建立的模型,小鼠胚胎干細(xì)胞在CollagenIV的刺激下能夠穩(wěn)定的分化成血管平滑肌細(xì)胞,并且隨著時間的推移,平滑肌標(biāo)志基因(SM-22α、SM-MHC、h1-Calponin)等表達(dá)增多。在此過程中,miR-214表達(dá)顯著上升趨勢。在功能研究實驗中,過表達(dá)miR-214能夠顯著促進(jìn)血管平滑肌細(xì)胞標(biāo)志基因在mRNA和蛋白水平的表達(dá),而抑制miR-214表達(dá)則降低平滑肌標(biāo)志基因的表達(dá)。多個miRNA的生物信息學(xué)分析數(shù)據(jù)庫分析軟件預(yù)測QKI是miR-214最有可能的的靶基因之一。在分化中的小鼠胚胎干細(xì)胞上,過表達(dá)miR-214能夠在mRNA和蛋白水平下調(diào)QKI表達(dá),而抑制miR-214引起QKI表達(dá)水平升高。熒光素酶基因報告實驗表明,過表達(dá)miR-214能夠顯著降低含有QKI3'UTR的熒光素酶活性,抑制miR-214能夠相應(yīng)引起QKI 3'UTR的熒光素酶活性在一定程度上升高。而對QKI 3'UTR中miR-214潛在結(jié)合位點進(jìn)行突變后,miR-214對熒光素酶活性的抑制作用消失。miR-214過表達(dá)模擬物和QKI過表達(dá)質(zhì)粒共同轉(zhuǎn)染細(xì)胞,發(fā)現(xiàn)高表達(dá)QKI可消除過表達(dá)的miR-214對小鼠胚胎干細(xì)胞向血管平滑肌細(xì)胞分化的促進(jìn)作用。同時,分別過表達(dá)和抑制QKI,發(fā)現(xiàn)過表達(dá)QKI能夠降低血管平滑肌細(xì)胞標(biāo)志基因的mRNA和蛋白表達(dá),抑制QKI表達(dá)則具有相反的作用。進(jìn)而,IP顯示,QKI能夠直接結(jié)合在平滑肌細(xì)胞分化關(guān)鍵轉(zhuǎn)錄因子的啟動子序列。最后,在成功建立的小鼠胚胎干細(xì)胞在體分化模型中,發(fā)現(xiàn)miR-214能夠促進(jìn)平滑肌分化標(biāo)志基因的表達(dá),在一定程度上從體內(nèi)實驗層面驗證了 miR-214促進(jìn)鼠胚胎干細(xì)胞向血管平滑肌細(xì)胞分化。結(jié)論:本研究成功揭示了 miR-214在胚胎干細(xì)胞向血管平滑肌細(xì)胞分化中的新功能。miR-214促進(jìn)胚胎干細(xì)胞向血管平滑肌細(xì)胞分化。QKI是miR-214的功能性靶基因,能夠直接結(jié)合在平滑肌分化關(guān)鍵轉(zhuǎn)錄因子的啟動子區(qū)域,從而抑制分化。miR-214直接結(jié)合QKI mRNA的3'UTR區(qū)域,下調(diào)QKI表達(dá),并通過調(diào)控QKI影響干細(xì)胞向血管平滑肌細(xì)胞分化。miR-214可能是防治平滑肌細(xì)胞相關(guān)血管損傷性疾病的新靶點。
[Abstract]:Research background and purpose: the basic pathological process of vascular injury with atherosclerosis as the basic histopathological changes is that vascular injury causes vascular repair and remodeling, and then the intima thickening of the vessels leads to the stenosis of the lumen. In this process, the vascular smooth muscle cells transfer, proliferate, migrate and stem cells through the phenotype. MicroRNA-214 (miR-214) has been found to regulate the functional and phenotypic transformation of mature vascular smooth muscle cells. However, the role of microRNA-214 in the differentiation of stem cells to vascular smooth muscle cells is not very clear. This study hopes to reveal the differentiation of miR-214 from embryonic stem cells to vascular smooth muscle cells. The purpose of this study is to identify the target gene of miR-214 and to elucidate its mechanism of action. Research methods: This study mainly used the stable, stable, in vitro mouse embryonic stem cells to differentiate into the smooth muscle cells, observe the changes in the expression of miR-214, and overexpress or inhibit the expression of miR-214, and study the smooth vascular smooth of the embryonic stem cells to the blood vessels. Secondly, the functional target genes of miR-214 were identified and confirmed by bioinformatics analysis and fluoropenzyme gene reporting system. Furthermore, a variety of molecular experimental techniques, such as immunoprecipitation (IP) and double luciferase gene report system, were used to prove that QKI was fine to the vascular smooth muscle of the embryonic stem cells. Finally, the effect of miR-214 on the differentiation of embryonic stem cells to smooth muscle cells in vivo was studied by using in vitro transfected cells to simulate the mouse embryonic stem cell differentiation model in vivo. Results: the mouse embryonic stem cells could be stimulated by CollagenIV. It is stable enough to differentiate into vascular smooth muscle cells, and the expression of the smooth muscle marker gene (SM-22, SM-MHC, h1-Calponin) increases with time. In this process, the expression of miR-214 is significantly increased. In the functional study, overexpression of miR-214 can significantly promote the level of vascular smooth muscle cell marker genes in mRNA and protein levels. The expression of miR-214 decreased the expression of the smooth muscle marker gene. The multi miRNA bioinformatics analysis database analysis software predicted that QKI was one of the most likely target genes of miR-214. In the differentiated mouse embryonic stem cells, the overexpression of miR-214 could reduce the QKI expression at the level of mRNA and protein, and inhibit miR-214. The expression level of QKI increased. The luciferase gene report showed that overexpression of miR-214 could significantly reduce the activity of luciferase containing QKI3'UTR, and the inhibition of miR-214 could increase the luciferase activity of QKI 3'UTR to a certain extent. While miR-214 latent at the binding site in QKI 3'UTR, miR-214 to fluorescein The inhibition of enzyme activity disappears.MiR-214 overexpressed and QKI overexpressed plasmids co transfected cells. It is found that high expression of QKI can eliminate the effect of overexpressed miR-214 on the differentiation of mouse embryonic stem cells to vascular smooth muscle cells. At the same time, the expression and inhibition of QKI, respectively, can be found to reduce the expression of QKI to reduce vascular smooth muscle cells. The mRNA and protein expression of the marker gene and the inhibition of the expression of QKI have the opposite effect. Then, IP shows that QKI can directly bind the promoter sequence of the key transcription factor of the smooth muscle cells. Finally, in the successfully established mouse embryonic stem cells in the body differentiation model, the expression of miR-214 can promote the expression of the marker gene of the smooth muscle differentiation. To a certain extent, the miR-214 promotes the differentiation of mouse embryonic stem cells to vascular smooth muscle cells. Conclusion: This study successfully revealed that the new function of miR-214 in the differentiation of embryonic stem cells to vascular smooth muscle cells (.MiR-214) promotes the differentiation of.QKI from embryonic stem cells to smooth muscle cells of the vascular smooth muscle cells, which is the function of miR-214. The target gene, which can directly bind to the promoter region of the key transcription factor of smooth muscle differentiation, inhibits the direct binding of the differentiated.MiR-214 to the 3'UTR region of the QKI mRNA, and down regulates the expression of QKI, and may be a new disease against vascular smooth muscle cells associated with vascular smooth muscle cell differentiation by regulating QKI to affect the differentiation of.MiR-214 into vascular smooth muscle cells. Target.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 汪雁歸;黃佳;楊天倫;;急性心肌梗死患者血清循環(huán)miR-214水平及其與冠狀動脈病變范圍的關(guān)系[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2015年04期

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本文編號：2109584