發(fā)布時(shí)間：2018-03-04 15:36

  本文選題：主動(dòng)脈瓣膜間質(zhì)細(xì)胞　切入點(diǎn)：成骨細(xì)胞分化　出處：《中南大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：分離并培養(yǎng)出人主動(dòng)脈瓣膜間質(zhì)細(xì)胞并利用鈣化培養(yǎng)基進(jìn)行干預(yù)建立其向成骨細(xì)胞分化的細(xì)胞模型 方法：收集sanford A型主動(dòng)脈夾層患者(夾層撕裂累及主動(dòng)脈瓣)的主動(dòng)脈瓣葉采用膠原酶消化法,分離出瓣膜間質(zhì)細(xì)胞。免疫組化鑒定細(xì)胞表型：平滑肌α肌動(dòng)蛋白,波形蛋白,CD31,平滑肌肌球蛋白和結(jié)蛋白。同時(shí)對(duì)細(xì)胞使用鈣化培養(yǎng)基(含β甘油磷酸鈉,地塞米松和維生素C)促進(jìn)主動(dòng)脈瓣膜間質(zhì)細(xì)胞向成骨細(xì)胞分化。通過(guò)堿性磷酸酶活性檢測(cè)、染色和鈣化結(jié)節(jié)染色(von Kossa法)以及western blot檢測(cè)堿性磷酸酶(ALP)及核結(jié)合因子α-1(Cbf a1)的表達(dá)評(píng)估該模型的效果 結(jié)果：分離出的主動(dòng)脈瓣膜間質(zhì)細(xì)胞能夠增殖,并表達(dá)平滑肌α肌動(dòng)蛋白和波形蛋白,但不表達(dá)CD31,平滑肌肌球蛋白和結(jié)蛋白。在鈣化培養(yǎng)基干預(yù)后的第21天,模型組較對(duì)照組的堿性磷酸酶活性、染色和鈣化結(jié)節(jié)染色更明顯,且表達(dá)更多的ALP和Cbfal。 結(jié)論：膠原酶消化法分離的人主動(dòng)脈瓣膜間質(zhì)細(xì)胞能夠在體外條件下增殖,并表達(dá)特有的表型,同時(shí)鈣化培養(yǎng)基能夠明顯地促進(jìn)主動(dòng)脈瓣膜間質(zhì)細(xì)胞向成骨細(xì)胞分化,ALP和Cbfal的表達(dá)并形成鈣化結(jié)節(jié)。 目的：證實(shí)內(nèi)皮素-1能夠促進(jìn)主動(dòng)脈瓣膜間質(zhì)細(xì)胞向成骨細(xì)胞分化,并討論內(nèi)皮素受體A (ETAR)和細(xì)胞外信號(hào)調(diào)節(jié)蛋白激酶(ERK)通路在其中扮演的角色。 方法：在鈣化培養(yǎng)基中分別添加不同濃度的內(nèi)皮素-1或內(nèi)皮素-1+ERK通路阻滯劑(PD98059)或內(nèi)皮素-1+ETAR阻滯劑(BQ-123)。在干預(yù)的第21天分別檢測(cè)堿性磷酸酶的活性和表達(dá)水平。干預(yù)的第21天檢測(cè)鈣化結(jié)節(jié),并檢測(cè)表型蛋白的變化。在培養(yǎng)基中添加內(nèi)皮素-1后,在不同時(shí)間(5min,10min,30min,60min,120min)檢測(cè)ERK通路的激活情況。在培養(yǎng)基中添加內(nèi)皮素-1和ETAR阻滯劑BQ-123和ERK通路阻滯劑PD98059,干預(yù)20分鐘后檢測(cè)ERK通路的激活程度。 結(jié)果：內(nèi)皮素-1以濃度依賴(lài)性促進(jìn)主動(dòng)脈瓣膜間質(zhì)細(xì)胞產(chǎn)生堿性磷酸酶,BQ-123及PD98059能夠明顯抑制內(nèi)皮素-1的上述作用。內(nèi)皮素-1可明顯促進(jìn)鈣化結(jié)節(jié)的產(chǎn)生及激活ERK通路,BQ-123和PD98059能夠抑制內(nèi)皮素-1對(duì)該通路的激活。 結(jié)論：內(nèi)皮素-1通過(guò)ETAR受體和ERK通路以濃度依賴(lài)性促進(jìn)主動(dòng)脈瓣膜間質(zhì)細(xì)胞向成骨細(xì)胞分化。
[Abstract]:Objective: to isolate and culture human aortic valve mesenchymal cells and to establish a cell model of osteoblast differentiation using calcification medium. Methods: the interstitial cells of aortic valve were isolated from aortic valve of sanford A aortic dissection by collagenase digestion. The phenotype of the cells was identified by immunohistochemistry: smooth muscle 偽 actin. Vimentin CD31, smooth muscle myosin and desmin. At the same time, calcification medium (containing 尾 -glycerophosphate, 尾 -glycerophosphate, 尾 -glycerophosphate) was used for cells. Dexamethasone and vitamin C) promote the differentiation of aortic valve interstitial cells into osteoblasts. The expression of alkaline phosphatase (ALP) and nuclear binding factor 偽 -1 (Cbfa1) were evaluated by staining and calcified nodule staining (von Kossa) and western blot to evaluate the effect of the model. Results: the isolated aortic valve stromal cells could proliferate and express smooth muscle 偽 actin and vimentin, but not CD31, smooth muscle myosin and desmin. The activity of alkaline phosphatase, staining and calcified nodule staining in model group were more obvious than those in control group, and the expression of ALP and Cbfalin were higher in the model group than in the control group. Conclusion: human aortic valve mesenchymal cells isolated by collagenase digestion can proliferate and express specific phenotypes in vitro. At the same time, calcification medium could obviously promote the expression of ALP and Cbfal in the differentiation of aortic valve interstitial cells into osteoblasts and form calcified nodules. Aim: to demonstrate that endothelin-1 can promote the differentiation of aortic valve interstitial cells into osteoblasts, and to discuss the roles of endothelin receptor A (ETAR) and extracellular signal regulated protein kinase (ERK) pathway in the differentiation of aortic valve interstitial cells into osteoblasts. Methods: different concentrations of endothelin-1 or endothelin-1 ERK pathway blockers (PD98059) or endothelin-1 ETAR blockers (BQ-123) were added to calcified media. The activity and expression of alkaline phosphatase were detected on the 21st day of intervention. ... on the 21st day of intervention, calcified nodules were detected, The changes of phenotypic protein were detected. The activation of ERK pathway was detected at different time points (5min / 10min / 30min / 60min / 120min). The activation of ERK pathway was detected by adding endothelin-1 and ETAR blocker BQ-123 and ERK pathway blocker PD98059. after 20 minutes of intervention, the activation of ERK pathway was detected. Results: endothelin-1 enhanced alkaline phosphatase production by aortic valve interstitial cells in a concentration-dependent manner. BQ-123 and PD98059 could significantly inhibit the above effects of endothelin-1. Endothelin-1 significantly promoted the production and activation of calcified nodules. ERK pathway BQ-123 and PD98059 can inhibit the activation of endothelin-1. Conclusion: endothelin-1 promotes the differentiation of aortic valve interstitial cells into osteoblasts in a concentration-dependent manner through ETAR receptor and ERK pathway.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329.2

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本文編號(hào)：1566288