本文選題：血管內(nèi)皮生長(zhǎng)因子 + 剪應(yīng)力�。� 參考：《第四軍醫(yī)大學(xué)》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(xì)胞或血管生成因子導(dǎo)入缺血組織中,促進(jìn)局部的血管新生和側(cè)枝循環(huán)形成,從而達(dá)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(xì)胞及如何獲得足夠數(shù)量的細(xì)胞以保證最佳的治療效果,干細(xì)胞是血管生成細(xì)胞重要的來源。 骨髓間充質(zhì)干細(xì)胞(MSCs)是一類存在于骨髓基質(zhì)內(nèi)的非造血干細(xì)胞來源的細(xì)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(diǎn),已經(jīng)成為構(gòu)建組織工程十分重要的種子細(xì)胞。MSCs生長(zhǎng)的生物化學(xué)環(huán)境和復(fù)雜的生物力學(xué)環(huán)境對(duì)其分化和表型表達(dá)有重要的影響。MSCs可以在體外擴(kuò)增并可經(jīng)不同條件誘導(dǎo)后分化為內(nèi)皮細(xì)胞、成骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞、肌細(xì)胞、神經(jīng)細(xì)胞等多種細(xì)胞。而內(nèi)皮細(xì)胞是導(dǎo)致血管新生的重要細(xì)胞,在體外血管內(nèi)皮生長(zhǎng)因子(VEGF)、堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、IGF-1、ECGS等細(xì)胞因子單獨(dú)或聯(lián)合的誘導(dǎo)下MSCs可以向內(nèi)皮細(xì)胞分化,使其成為一種可能用于治療性血管新生的細(xì)胞來源。此外,因其體外可大量擴(kuò)增,有望解決血管內(nèi)皮細(xì)胞來源不足的問題。但各種細(xì)胞因子普遍價(jià)格昂貴,以及潛在的細(xì)胞生物安全性問題未得到解決。而相對(duì)細(xì)胞因子的化學(xué)環(huán)境,生物力學(xué)環(huán)境對(duì)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(xì)胞成熟并保持功能的不可缺少的促進(jìn)因素,已有研究表明[8],剪應(yīng)力可以誘導(dǎo)了胚胎干細(xì)胞和內(nèi)皮祖細(xì)胞兩類干細(xì)胞向內(nèi)皮細(xì)胞分化。因此提示剪應(yīng)力也可能誘導(dǎo)MSCs向內(nèi)皮細(xì)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(duì)人MSCs向內(nèi)皮細(xì)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(xì)胞形態(tài)。3.用免疫組織化學(xué)法檢測(cè)細(xì)胞表面標(biāo)記物CD44對(duì)MSCs進(jìn)行鑒定。 第二部分VEGF對(duì)MSCs向內(nèi)皮細(xì)胞分化的影響將培養(yǎng)得到的MSCs分為對(duì)照組和VEGF誘導(dǎo)組,誘導(dǎo)組給予10μg/L VEGF作用。分別于誘導(dǎo)24小時(shí)(h)、7天(d)后觀察細(xì)胞形態(tài)變化,采用間接免疫熒光染色法檢測(cè)內(nèi)皮細(xì)胞標(biāo)記物Von Willebrand factor (vWF)的表達(dá)情況,用Dil標(biāo)記的乙�；疞DL(Dil-Ac-LDL)攝取實(shí)驗(yàn)檢測(cè)內(nèi)皮細(xì)胞脂質(zhì)攝取功能。 第三部分剪應(yīng)力對(duì)MSCs向內(nèi)皮細(xì)胞分化的影響將培養(yǎng)得到的MSCs分為對(duì)照組和剪應(yīng)力誘導(dǎo)組(8、15 dyn/cm2),誘導(dǎo)組在平行平板流動(dòng)腔裝置中施加剪應(yīng)力。誘導(dǎo)24h后觀察細(xì)胞形態(tài)變化、vWF表達(dá)情況以及Dil-Ac-LDL攝取功能。 第四部分剪應(yīng)力聯(lián)合VEGF對(duì)MSCs向內(nèi)皮細(xì)胞分化的影響將培養(yǎng)得到的MSCs分為對(duì)照組、剪應(yīng)力聯(lián)合誘導(dǎo)組(8、15 dyn/cm2)與VEGF(10μg/L)聯(lián)合誘導(dǎo)組。誘導(dǎo)24h后觀察細(xì)胞形態(tài)變化, vWF表達(dá)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(zhǎng)梭形,免疫組化染色顯示CD44陽性,證明培養(yǎng)細(xì)胞為MSCs。 2.與對(duì)照組相比,VEGF誘導(dǎo)24h后MSCs形態(tài)無明顯變化,vWF染色陰性,Dil-Ac-LDL攝取實(shí)驗(yàn)陰性。VEGF誘導(dǎo)7d后MSCs形態(tài)呈扁圓或多角形,類似內(nèi)皮細(xì)胞,vWF染色陽性,Dil-Ac-LDL攝取實(shí)驗(yàn)陽性,提示MSCs已分化為內(nèi)皮細(xì)胞。 3.與對(duì)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(shí)驗(yàn)陽性,提示MSCs已分化為內(nèi)皮細(xì)胞。而15dyn/cm2剪應(yīng)力作用24h后,vWF染色與Dil-Ac-LDL攝取實(shí)驗(yàn)均陰性,提示MSCs未向內(nèi)皮細(xì)胞分化。 4.與單獨(dú)剪應(yīng)力作用組相比,剪應(yīng)力與VEGF聯(lián)合作用24h后,細(xì)胞密度明顯增大,vWF染色強(qiáng)陽性,Dil-Ac-LDL攝取實(shí)驗(yàn)陽性,提示MSCs不僅分化為內(nèi)皮細(xì)胞,而且發(fā)生增殖。 結(jié)論 1.經(jīng)觀察細(xì)胞形態(tài)和細(xì)胞表面標(biāo)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(dǎo)MSCs向內(nèi)皮細(xì)胞分化。 3. 8dyn/cm2剪應(yīng)力可誘導(dǎo)MSCs開始向內(nèi)皮細(xì)胞分化,而增至15dyn/cm2剪應(yīng)力時(shí)此作用消失。 4.剪應(yīng)力聯(lián)合VEGF不僅可誘導(dǎo)MSCs向內(nèi)皮細(xì)胞分化,而且可使細(xì)胞密度增大,誘導(dǎo)效果優(yōu)于單獨(dú)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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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本文編號(hào)：1817289