本文選題：血管內(nèi)皮生長因子 + 剪應(yīng)力��； 參考：《第四軍醫(yī)大學》2009年碩士論文

【摘要】： 研究背景 治療性血管新生即將外源性的血管生成細胞或血管生成因子導入缺血組織中,促進局部的血管新生和側(cè)枝循環(huán)形成,從而達到治療缺血性疾病的目的,是近年來提出的治療缺血性疾病特別是缺血性心臟病的新概念。目前的研究主要集中于選擇何種血管生成細胞及如何獲得足夠數(shù)量的細胞以保證最佳的治療效果,干細胞是血管生成細胞重要的來源。 骨髓間充質(zhì)干細胞(MSCs)是一類存在于骨髓基質(zhì)內(nèi)的非造血干細胞來源的細胞亞群。MSCs具有容易分離培養(yǎng)、多向分化能力、免疫排斥反應(yīng)小的特點,已經(jīng)成為構(gòu)建組織工程十分重要的種子細胞。MSCs生長的生物化學環(huán)境和復雜的生物力學環(huán)境對其分化和表型表達有重要的影響。MSCs可以在體外擴增并可經(jīng)不同條件誘導后分化為內(nèi)皮細胞、成骨細胞、軟骨細胞、脂肪細胞、肌細胞、神經(jīng)細胞等多種細胞。而內(nèi)皮細胞是導致血管新生的重要細胞,在體外血管內(nèi)皮生長因子(VEGF)、堿性成纖維細胞生長因子(bFGF)、IGF-1、ECGS等細胞因子單獨或聯(lián)合的誘導下MSCs可以向內(nèi)皮細胞分化,使其成為一種可能用于治療性血管新生的細胞來源。此外,因其體外可大量擴增,有望解決血管內(nèi)皮細胞來源不足的問題。但各種細胞因子普遍價格昂貴,以及潛在的細胞生物安全性問題未得到解決。而相對細胞因子的化學環(huán)境,生物力學環(huán)境對MSCs影響的研究仍處于起步階段。血流剪應(yīng)力是血管內(nèi)皮細胞成熟并保持功能的不可缺少的促進因素,已有研究表明[8],剪應(yīng)力可以誘導了胚胎干細胞和內(nèi)皮祖細胞兩類干細胞向內(nèi)皮細胞分化。因此提示剪應(yīng)力也可能誘導MSCs向內(nèi)皮細胞分化。 目的 本研究擬探討在體外培養(yǎng)的條件下,VEGF和剪應(yīng)力對人MSCs向內(nèi)皮細胞分化的影響。 方法 第一部分MSCs的分離培養(yǎng)和鑒定1.取健康成人骨髓,密度梯度離心結(jié)合貼壁法分離MSCs,體外培養(yǎng)并傳代。2.光鏡下觀察細胞形態(tài)。3.用免疫組織化學法檢測細胞表面標記物CD44對MSCs進行鑒定。 第二部分VEGF對MSCs向內(nèi)皮細胞分化的影響將培養(yǎng)得到的MSCs分為對照組和VEGF誘導組,誘導組給予10μg/L VEGF作用。分別于誘導24小時(h)、7天(d)后觀察細胞形態(tài)變化,采用間接免疫熒光染色法檢測內(nèi)皮細胞標記物Von Willebrand factor (vWF)的表達情況,用Dil標記的乙�；疞DL(Dil-Ac-LDL)攝取實驗檢測內(nèi)皮細胞脂質(zhì)攝取功能。 第三部分剪應(yīng)力對MSCs向內(nèi)皮細胞分化的影響將培養(yǎng)得到的MSCs分為對照組和剪應(yīng)力誘導組(8、15 dyn/cm2),誘導組在平行平板流動腔裝置中施加剪應(yīng)力。誘導24h后觀察細胞形態(tài)變化、vWF表達情況以及Dil-Ac-LDL攝取功能。 第四部分剪應(yīng)力聯(lián)合VEGF對MSCs向內(nèi)皮細胞分化的影響將培養(yǎng)得到的MSCs分為對照組、剪應(yīng)力聯(lián)合誘導組(8、15 dyn/cm2)與VEGF(10μg/L)聯(lián)合誘導組。誘導24h后觀察細胞形態(tài)變化, vWF表達情況以及Dil-Ac-LDL攝取功能。 結(jié)果 1.分離培養(yǎng)得到的MSCs形態(tài)呈長梭形,免疫組化染色顯示CD44陽性,證明培養(yǎng)細胞為MSCs。 2.與對照組相比,VEGF誘導24h后MSCs形態(tài)無明顯變化,vWF染色陰性,Dil-Ac-LDL攝取實驗陰性。VEGF誘導7d后MSCs形態(tài)呈扁圓或多角形,類似內(nèi)皮細胞,vWF染色陽性,Dil-Ac-LDL攝取實驗陽性,提示MSCs已分化為內(nèi)皮細胞。 3.與對照組相比,8dyn/cm2剪應(yīng)力作用24h后,MSCs呈扁圓或多角形,vWF染色弱陽性,Dil-Ac-LDL攝取實驗陽性,提示MSCs已分化為內(nèi)皮細胞。而15dyn/cm2剪應(yīng)力作用24h后,vWF染色與Dil-Ac-LDL攝取實驗均陰性,提示MSCs未向內(nèi)皮細胞分化。 4.與單獨剪應(yīng)力作用組相比,剪應(yīng)力與VEGF聯(lián)合作用24h后,細胞密度明顯增大,vWF染色強陽性,Dil-Ac-LDL攝取實驗陽性,提示MSCs不僅分化為內(nèi)皮細胞,而且發(fā)生增殖。 結(jié)論 1.經(jīng)觀察細胞形態(tài)和細胞表面標志物鑒定,采用密度梯度離心結(jié)合貼壁法可在體外成功分離、培養(yǎng)MSCs。 2.VEGF可誘導MSCs向內(nèi)皮細胞分化。 3. 8dyn/cm2剪應(yīng)力可誘導MSCs開始向內(nèi)皮細胞分化,而增至15dyn/cm2剪應(yīng)力時此作用消失。 4.剪應(yīng)力聯(lián)合VEGF不僅可誘導MSCs向內(nèi)皮細胞分化,而且可使細胞密度增大,誘導效果優(yōu)于單獨使用剪應(yīng)力。
[Abstract]:Background of the study



The present study focuses on the choice of angiogenesis cells and how to obtain a sufficient number of cells to ensure optimal therapeutic effects , and stem cells are important sources of angiogenesis cells .



Bone marrow mesenchymal stem cells ( MSCs ) are a kind of non - hematopoietic stem cells derived from bone marrow stromal cells . MSCs can be differentiated into endothelial cells , osteoblasts , chondrocytes , fat cells , muscle cells , nerve cells and so on .



Purpose



This study intends to explore the effect of VEGF and shear stress on the differentiation of human MSCs into endothelial cells under the condition of in vitro culture .



method



MSCs were isolated and cultured in vitro . MSCs were isolated from healthy adult bone marrow and density gradient centrifugation combined with adherent method . The cells were cultured in vitro and passaged .



In the second part , the effect of VEGF on the differentiation of MSCs into endothelial cells was divided into control group and VEGF - induced group . After 24 hours ( h ) and 7 days ( d ) , the morphological changes of endothelial cells were observed .



The effect of the third part of shear stress on the differentiation of MSCs into endothelial cells was divided into control group and shear stress inducing group ( 8 , 15 dy/ cm2 ) , and the induced group applied shear stress in the parallel plate flow cavity device . After 24 h , the morphological changes of the cells , the expression of VWF and the Dil - Ac - LDL uptake function were observed .



In the fourth part , the effect of shear stress combined with VEGF on the differentiation of MSCs into endothelial cells was divided into two groups : control group , shear stress combination induction group ( 8 , 15 dy/ cm2 ) and VEGF ( 10 渭g / L ) . After 24 hours of induction , the morphological changes of cells , the expression of VWF and Dil - Ac - LDL uptake were observed .



Results



1 . The morphology of MSCs isolated from culture was spindle - shaped . Immunohistochemical staining showed CD44 positive , and proved that the cultured cells were MSCs .



2 . Compared with the control group , there was no obvious change in the morphology of MSCs after 24 hours of VEGF induction , and the negative result of Dil - Ac - LDL uptake was negative . After 7 days of VEGF - induced MSCs , MSCs appeared flat or polygonal , similar to endothelial cells , and the positive of Dil - Ac - LDL uptake were positive , suggesting that MSCs were differentiated into endothelial cells .



3 . Compared with the control group , MSCs were stained weakly positive and Dil - Ac - LDL uptake was positive after 24 h of shear stress , which showed that MSCs were differentiated into endothelial cells .



4 . Compared with the single shear stress group , the density of the cells increased obviously after 24 h combined with VEGF , and the density of the cells was significantly increased , and the Dil - Ac - LDL uptake was positive , suggesting that MSCs were not only differentiated into endothelial cells , but also proliferated .



Conclusion



1 . Through the observation of cell morphology and cell surface marker identification , the MSCs can be isolated and cultured in vitro by density gradient centrifugation combined with adherent method .



2 . VEGF induced MSCs to differentiate into endothelial cells .



3 . The shear stress of 3 . 8 ( 3 . 8 ) / cm ~ 2 can induce the differentiation of MSCs to endothelial cells , and this effect disappears when the shear stress increases to 15 % / cm & lt ; 2 & gt ; / cm & lt ; 2 & gt ; .



4 . The shear stress combined with VEGF not only can induce MSCs to differentiate into endothelial cells , but also increase the density of cells , which is superior to the use of shear stress alone .

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前3條

1 馮濱,劉迎龍,馮凱,龔茹,陳虎;人骨髓間質(zhì)干細胞體外擴增和向內(nèi)皮細胞定向誘導分化的研究[J];中國病理生理雜志;2005年08期

2 王建安;謝小潔;;干細胞移植治療擴張型心肌病[J];中華心血管病雜志;2006年03期

3 晉軍,黃嵐,祝善俊,向常青,李洪,耿建萌,吳旭;VEGF在大鼠心肌梗死急性期表達的意義及蛻皮甾酮的促側(cè)支循環(huán)作用[J];中國介入心臟病學雜志;2002年03期

，

本文編號：1817289