本文選題：血管緊張素Ⅱ + 心外膜祖細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目前,冠心病仍是世界上最常見的死亡原因,嚴(yán)重威脅人類的健康。研究冠狀動脈的發(fā)育,對于冠心病的防治以及人類的健康保障具有重要意義。心外膜祖細(xì)胞(epicardial progenitor cells;Epi Cs)是最近提出的一組心臟祖細(xì)胞,起源于前體心外膜祖細(xì)胞,是一個表達(dá)Tbx18、Wt1、Tcf21等多種標(biāo)記基因的祖細(xì)胞池。因其可分化形成血管平滑肌細(xì)胞、成纖維細(xì)胞、竇房結(jié)起搏細(xì)胞等多種心系細(xì)胞,對胚胎心臟的形成起重要作用,成為目前心肌再生研究的一個焦點。目前已有大量研究證實心外膜祖細(xì)胞是冠狀動脈血管平滑肌細(xì)胞的主要來源之一,且參與該分化過程的機(jī)制眾多。血管緊張素Ⅱ(Angiontensin II;Ang II)作為腎素-血管緊張素-醛固酮系統(tǒng)中最重要的效應(yīng)分子,主要通過Ang II受體AT1、AT2起作用,除了本身固有的體液調(diào)節(jié)功能外,還是一個生長調(diào)節(jié)因子,與細(xì)胞的生長、發(fā)育、分化及調(diào)亡均密切相關(guān)。除此之外,大量研究發(fā)現(xiàn)AngⅡ可以誘導(dǎo)脂肪干細(xì)胞、骨髓間充質(zhì)干細(xì)胞、等分化為平滑肌細(xì)胞。因此明確AngⅡ?qū)π耐饽ぷ婕?xì)胞向血管平滑肌細(xì)胞分化的作用具有重要的臨床意義。第一部分:心外膜祖細(xì)胞的培養(yǎng)及鑒定目的:在體外成功培養(yǎng)C57BL/6小鼠E12.5d胚胎心外膜祖細(xì)胞,為體外研究心外膜祖細(xì)胞向血管平滑肌細(xì)胞的分化機(jī)制奠定基礎(chǔ)。方法:分離小鼠E12.5d胚胎心臟組織進(jìn)行原代細(xì)胞培養(yǎng)得到心外膜祖細(xì)胞,并通過細(xì)胞免疫熒光方法檢測Epi Cs細(xì)胞特異性標(biāo)志物Tbx18、Wt1的表達(dá)。結(jié)果:所培養(yǎng)的原代細(xì)胞高度表達(dá)Tbx18、Wt1,結(jié)合其來源和形態(tài)證實成功培養(yǎng)出心外膜祖細(xì)胞。結(jié)論:利用C57BL/6小鼠E12.5d胚胎心臟組織能成功培養(yǎng)出心外膜祖細(xì)胞。第二部分:Ang II通過受體AT1誘導(dǎo)心外膜祖細(xì)胞向血管平滑肌細(xì)胞的分化目的:探討Ang II誘導(dǎo)心外膜祖細(xì)胞向血管平滑肌細(xì)胞的分化及相關(guān)通路。方法:細(xì)胞免疫熒光檢測心外膜祖細(xì)胞表面AngⅡ作用受體AT1、AT2的表達(dá)。培養(yǎng)得到的原代心外膜祖細(xì)胞隨機(jī)分為AngⅡ組、AngⅡ+氯沙坦組、AngⅡ+PD123319組和對照組進(jìn)行干預(yù)。免疫熒光檢測血管平滑肌細(xì)胞特異性標(biāo)志物α-平滑肌肌動蛋白(alpha-sooth muscle actin,α-SMA)、肌球蛋白重鏈11(Myosin heavy chain 11,Myh11)在各組的表達(dá)情況,實時熒光定量PCR檢測α-SMA、Myh11在各組的m RNA表達(dá)水平,膠原凝膠收縮實驗分別檢測干預(yù)后細(xì)胞的收縮功能。結(jié)果:心外膜祖細(xì)胞膜表面有AngⅡ受體AT1、AT2的表達(dá),給予AngⅡ干預(yù)7天后實驗組α-SMA、Myh11的表達(dá)明顯增多,其m RNA水平明顯高于對照組,經(jīng)AngⅡ誘導(dǎo)的細(xì)胞在卡巴膽堿的作用下也展示了明顯的收縮性。氯沙坦阻斷AngⅡ受體AT1后,AngⅡ干預(yù)使心外膜祖細(xì)胞的α-SMA、Myh11表達(dá)下降;PD123319阻斷AngⅡ受體AT2后,AngⅡ干預(yù)對α-SMA、Myh11表達(dá)沒有明顯影響。結(jié)論:血管緊張素Ⅱ可以通過受體AT1有效誘導(dǎo)小鼠心外膜祖細(xì)胞分化為血管平滑肌細(xì)胞。
[Abstract]:At present, coronary heart disease is still the most common cause of death in the world, a serious threat to human health. It is of great significance to study the development of coronary artery for prevention and treatment of coronary heart disease and human health protection. Epicardial progenitor cell (epicardial progenitor cells) is a recently proposed group of cardiac progenitor cells, originated from precursor epicardial progenitor cells. It is a pool of progenitor cells expressing Tbx18, Wt1, Tcf21 and other marker genes. Because of its differentiation into vascular smooth muscle cells, fibroblasts, sinus node pacemaker cells and other heart cells, it plays an important role in the formation of embryonic heart, and has become a focus of myocardial regeneration. At present, many studies have confirmed that epicardial progenitor cells are one of the main sources of vascular smooth muscle cells in coronary artery, and there are many mechanisms involved in the differentiation process. As the most important effector of renin-angiotensin-aldosterone system, angiotensin II (Angiontensin II) acts mainly through Ang II receptor AT1, AT2, and is also a growth regulator in addition to its inherent humoral regulatory function. It is closely related to cell growth, development, differentiation and apoptosis. In addition, a large number of studies have found that Ang 鈪，

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