發(fā)布時間：2018-05-12 05:14

  本文選題：血管緊張素Ⅱ + 心外膜祖細胞��； 參考：《重慶醫(yī)科大學》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。心外膜祖細胞(epicardial progenitor cells;Epi Cs)是最近提出的一組心臟祖細胞,起源于前體心外膜祖細胞,是一個表達Tbx18、Wt1、Tcf21等多種標記基因的祖細胞池。因其可分化形成血管平滑肌細胞、成纖維細胞、竇房結起搏細胞等多種心系細胞,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胞是冠狀動脈血管平滑肌細胞的主要來源之一,且參與該分化過程的機制眾多。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節(jié)功能外,還是一個生長調節(jié)因子,與細胞的生長、發(fā)育、分化及調亡均密切相關。除此之外,大量研究發(fā)現(xiàn)AngⅡ可以誘導脂肪干細胞、骨髓間充質干細胞、等分化為平滑肌細胞。因此明確AngⅡ對心外膜祖細胞向血管平滑肌細胞分化的作用具有重要的臨床意義。第一部分:心外膜祖細胞的培養(yǎng)及鑒定目的:在體外成功培養(yǎng)C57BL/6小鼠E12.5d胚胎心外膜祖細胞,為體外研究心外膜祖細胞向血管平滑肌細胞的分化機制奠定基礎。方法:分離小鼠E12.5d胚胎心臟組織進行原代細胞培養(yǎng)得到心外膜祖細胞,并通過細胞免疫熒光方法檢測Epi Cs細胞特異性標志物Tbx18、Wt1的表達。結果:所培養(yǎng)的原代細胞高度表達Tbx18、Wt1,結合其來源和形態(tài)證實成功培養(yǎng)出心外膜祖細胞。結論:利用C57BL/6小鼠E12.5d胚胎心臟組織能成功培養(yǎng)出心外膜祖細胞。第二部分:Ang II通過受體AT1誘導心外膜祖細胞向血管平滑肌細胞的分化目的:探討Ang II誘導心外膜祖細胞向血管平滑肌細胞的分化及相關通路。方法:細胞免疫熒光檢測心外膜祖細胞表面AngⅡ作用受體AT1、AT2的表達。培養(yǎng)得到的原代心外膜祖細胞隨機分為AngⅡ組、AngⅡ+氯沙坦組、AngⅡ+PD123319組和對照組進行干預。免疫熒光檢測血管平滑肌細胞特異性標志物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)在各組的表達情況,實時熒光定量PCR檢測α-SMA、Myh11在各組的m RNA表達水平,膠原凝膠收縮實驗分別檢測干預后細胞的收縮功能。結果:心外膜祖細胞膜表面有AngⅡ受體AT1、AT2的表達,給予AngⅡ干預7天后實驗組α-SMA、Myh11的表達明顯增多,其m RNA水平明顯高于對照組,經(jīng)AngⅡ誘導的細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干預使心外膜祖細胞的α-SMA、Myh11表達下降;PD123319阻斷AngⅡ受體AT2后,AngⅡ干預對α-SMA、Myh11表達沒有明顯影響。結論:血管緊張素Ⅱ可以通過受體AT1有效誘導小鼠心外膜祖細胞分化為血管平滑肌細胞。
[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，

本文編號：1877247