剪应力和血管内皮生长因子对人骨髓间充质干细胞向血管内皮细胞分化的影响

发布时间：2018-04-28 23:07

  本文选题：血管内皮生长因子 + 剪应力　； 参考：《第四军医大学》2009年硕士论文

【摘要】： 研究背景 治疗性血管新生即将外源性的血管生成细胞或血管生成因子导入缺血组织中,促进局部的血管新生和侧枝循环形成,从而达到治疗缺血性疾病的目的,是近年来提出的治疗缺血性疾病特别是缺血性心脏病的新概念。目前的研究主要集中于选择何种血管生成细胞及如何获得足够数量的细胞以保证最佳的治疗效果,干细胞是血管生成细胞重要的来源。 骨髓间充质干细胞(MSCs)是一类存在于骨髓基质内的非造血干细胞来源的细胞亚群。MSCs具有容易分离培养、多向分化能力、免疫排斥反应小的特点,已经成为构建组织工程十分重要的种子细胞。MSCs生长的生物化学环境和复杂的生物力学环境对其分化和表型表达有重要的影响。MSCs可以在体外扩增并可经不同条件诱导后分化为内皮细胞、成骨细胞、软骨细胞、脂肪细胞、肌细胞、神经细胞等多种细胞。而内皮细胞是导致血管新生的重要细胞,在体外血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、IGF-1、ECGS等细胞因子单独或联合的诱导下MSCs可以向内皮细胞分化,使其成为一种可能用于治疗性血管新生的细胞来源。此外,因其体外可大量扩增,有望解决血管内皮细胞来源不足的问题。但各种细胞因子普遍价格昂贵,以及潜在的细胞生物安全性问题未得到解决。而相对细胞因子的化学环境,生物力学环境对MSCs影响的研究仍处于起步阶段。血流剪应力是血管内皮细胞成熟并保持功能的不可缺少的促进因素,已有研究表明[8],剪应力可以诱导了胚胎干细胞和内皮祖细胞两类干细胞向内皮细胞分化。因此提示剪应力也可能诱导MSCs向内皮细胞分化。 目的 本研究拟探讨在体外培养的条件下,VEGF和剪应力对人MSCs向内皮细胞分化的影响。 方法 第一部分MSCs的分离培养和鉴定1.取健康成人骨髓,密度梯度离心结合贴壁法分离MSCs,体外培养并传代。2.光镜下观察细胞形态。3.用免疫组织化学法检测细胞表面标记物CD44对MSCs进行鉴定。 第二部分VEGF对MSCs向内皮细胞分化的影响将培养得到的MSCs分为对照组和VEGF诱导组,诱导组给予10μg/L VEGF作用。分别于诱导24小时(h)、7天(d)后观察细胞形态变化,采用间接免疫荧光染色法检测内皮细胞标记物Von Willebrand factor (vWF)的表达情况,用Dil标记的乙酰化LDL(Dil-Ac-LDL)摄取实验检测内皮细胞脂质摄取功能。 第三部分剪应力对MSCs向内皮细胞分化的影响将培养得到的MSCs分为对照组和剪应力诱导组(8、15 dyn/cm2),诱导组在平行平板流动腔装置中施加剪应力。诱导24h后观察细胞形态变化、vWF表达情况以及Dil-Ac-LDL摄取功能。 第四部分剪应力联合VEGF对MSCs向内皮细胞分化的影响将培养得到的MSCs分为对照组、剪应力联合诱导组(8、15 dyn/cm2)与VEGF(10μg/L)联合诱导组。诱导24h后观察细胞形态变化, vWF表达情况以及Dil-Ac-LDL摄取功能。 结果 1.分离培养得到的MSCs形态呈长梭形,免疫组化染色显示CD44阳性,证明培养细胞为MSCs。 2.与对照组相比,VEGF诱导24h后MSCs形态无明显变化,vWF染色阴性,Dil-Ac-LDL摄取实验阴性。VEGF诱导7d后MSCs形态呈扁圆或多角形,类似内皮细胞,vWF染色阳性,Dil-Ac-LDL摄取实验阳性,提示MSCs已分化为内皮细胞。 3.与对照组相比,8dyn/cm2剪应力作用24h后,MSCs呈扁圆或多角形,vWF染色弱阳性,Dil-Ac-LDL摄取实验阳性,提示MSCs已分化为内皮细胞。而15dyn/cm2剪应力作用24h后,vWF染色与Dil-Ac-LDL摄取实验均阴性,提示MSCs未向内皮细胞分化。 4.与单独剪应力作用组相比,剪应力与VEGF联合作用24h后,细胞密度明显增大,vWF染色强阳性,Dil-Ac-LDL摄取实验阳性,提示MSCs不仅分化为内皮细胞,而且发生增殖。 结论 1.经观察细胞形态和细胞表面标志物鉴定,采用密度梯度离心结合贴壁法可在体外成功分离、培养MSCs。 2.VEGF可诱导MSCs向内皮细胞分化。 3. 8dyn/cm2剪应力可诱导MSCs开始向内皮细胞分化,而增至15dyn/cm2剪应力时此作用消失。 4.剪应力联合VEGF不仅可诱导MSCs向内皮细胞分化,而且可使细胞密度增大,诱导效果优于单独使用剪应力。
[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 .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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