不同型式流体切应力对血管内皮细胞生理生化的影响

血管内皮细胞经常处于血液流动力的作用下,它能感知血流力的变化,在调节血管的功能和结构中起状重要作用。血液流动力的变化与某些血客性疾病的发生发展有着密切的关系,因此血液流动力对内皮细胞的影响受到广泛关注。本文就流体切应力对内皮细胞生理、生化影响的研究进展作一简要综述。     

流体切应力对血管内皮细胞的作用

20世纪80年代生物医学科学领域出现了一个新的热点一血管内皮细胞的形态和机能的研究。不仅因为血管内皮细胞的异常与心脑血管病、肿瘤、免疫性疾病、出血、辐射、移植排斥反应等等重要疾病发病和防治有关,而且和组织工程、人工器官等的开发有着密切关系。所以该领域一开始就吸引着国内外很多学者的兴趣和关注,该领域的炽热状态可能会维持到下世纪中叶。一、血管内皮细胞的形态和功能内皮细胞（endothelialcell,EC）是衬贴在心血管内脏面的单层鳞状细胞。厚约0．1－1μm,大小为（10－50）μm×（25－40）μm,多角形,全身血管内皮细胞…     

流体切应力对血管内皮细胞粘附分子ICAM-1的影响

目的 :探讨血液流动产生的切应力对白细胞 血管内皮细胞 (VEC)相互粘附所产生的影响及其在炎症、免疫反应、动脉粥样硬化发生发展中所起的重要作用。方法 :将已汇合的人脐静脉内皮细胞玻片放于平板流动腔中 ,以层流不同切应力 0 .6、1.2、2 .4Pa剪切不同时间 (2、8、12h) ,用免疫细胞化学方法检测VEC表面ICAM 1表达 ,以显微镜下计数阳性细胞率表示。结果 :稳层流切应力能上调HUVECICAM 1表达 ,切应力是内皮细胞粘附分子表达的调节因素之一。     

流体切应力对血管内皮细胞基因表达的影响

血液产生的切应力可以引起血管中的内皮细胞发生生物物理、生化和基因调控水平上的反应,从而调节内皮的结构和功能。当内皮细胞受到的切应力发生变化时,即可引起细胞基因表达上的变化,从而可能引发一些常见的心血管疾病。对切应力调控内皮细胞基因表达机制的研究,有助于找到治疗以上疾病的临床方法。综述了切应力对内皮细胞基因表达的影响及其调控机制,并对其在临床治疗、小口径人工血管构建和药物设计等方面的应用前景进行了展望。     

流体切应力对血管内皮细胞的生物学作用

生物医学技术的不断发展，为心血管人工移植物(如人工血管、人工瓣膜)的临床应用提供了广阔的发展空间。内皮细胞与平滑肌细胞作为血管壁的主要组成细胞在人工血管的构建研究中占有重要地位。体外培养是研究内皮细胞与平滑肌细胞细胞形态和功能的重要手段。但静态条件下的内皮细胞与平滑肌细胞的联合培养缺少在体条件下的力学环境，因而近年来国内外开始将两种研究方法结合起来，研究在力学环境下内皮细胞与平滑肌细胞的相互作用，从而为人工血管移植物的研究提供理论基础。     

流体切应力梯度对血管内皮细胞排列和形状的影响

目的研究不同梯度切应力作用下,血管内皮细胞(endothelial cells,ECs)排列和形状变化,旨在了解流体切应力梯度对ECs形态的影响,为进一步探讨其功能变化提供实验基础。方法建立可对体外培养ECs施加梯度切应力的流动腔装置,并应用该装置对人脐静脉ECs加载了大小在15dyn/cm2～6.6dyn/cm2(1dyn=10-5N)范围、梯度分别为1.5dyn/cm2和3dyn/cm2的切应力,加载时间均为6h。比较这两种不同切应力梯度对ECs的细胞方向角、细胞宽长比和细胞形态指数的影响。结果在不同切应力梯度作用下,ECs的细胞方向角分布散乱,细胞无排列规律。与3dyn/cm2相比,1.5dyn/cm2切应力梯度下ECs的宽长比和细胞形态指数明显减少,趋向于拉伸状态。结论在不同切应力梯度作用下,ECs均排列紊乱,无规律可循。然而,在相对较小的切应力梯度作用下,细胞容易被拉伸,细胞形状趋向于伸长,而较大切应力梯度作用下,细胞形状则趋向于圆形。     

流体切应力对人内皮细胞迁移和整合素基因表达的影响

目的研究流体切应力空间梯度和均匀切应力对内皮细胞（ECs）迁移和整合素基因表达的影响，为阐明应力诱导血管重建的分子机制提供一些实验依据。方法将脐静脉内皮细胞置于平行平板流动腔系统，分别施加11dyn／cm^2的均匀切应力（矩形组）和5～14dyn／cm^2的切应力梯度（梯度组）为受力组，以静态条件下培养的ECs为对照组（静止组）。Transwell方法检测切应力对ECs迁移能力的影响；半定量RT-PCR测定ECs整合素α3β1、α5β1mRNA3、6和12h的表达情况。结果①同静止组相比，切应力梯度组ECs3h的迁移（88±4 vs 23±3，P〈0．01，n=3）能力显著提高；而均匀切应力组ECs3h迁移（21±3VS23±3）同静止组相比，差异无统计学意义（P〉0．05，n=3）；②ECs受切应力梯度作用3h即可诱导ECs整合素α3β1、α5β1mRNA表达（P〈0．01，P〈0．05）；均匀切应力于6h激活ECs整合素理，亚型表达（P〈0．01），α3 、β1亚型表达延迟到12h激活（P〈0．01）。结论切应力通过上调整合素基因表达促进了ECs迁移，提示整合素信号通路参与了切应力条件下ECs迁移过程的信号转导。     

应力培养对血管内皮细胞形态结构的影响

为在体外形成一个抗流动切应力作用能力强的单层内皮细胞，在１５ｄｙｎｅｓ／ｃｍ＾２切应力的条件下，培养内皮细胞２４ｈ。细胞内皮细胞中央出现应力纤维，Ｇ０／Ｇ１期细胞增加，这一现象提示，在切应力条件下培养内皮细胞，可增强细胞抗血流冲击的能力。     

血流切应力对血管内皮细胞PCP信号通路与初级纤毛发生的影响

目的研究不同血流切应力(fluid shear stress, FSS)对内皮细胞平面细胞极性通路(planar cell polarity, PCP)的调节作用,进一步探讨FSS、PCP信号通路以及纤毛发生之间的关系。方法建立可调控FSS的流体动力学细胞培养模型,qPCR以及免疫荧光检测不同FSS作用下PCP信号通路核心蛋白Dvl2及纤毛装配蛋白IFT88的mRNA表达与细胞定位以及两者的共定位,Western bolt (WB)检测不同FSS作用18 h时Dvl2蛋白表达。结果 qPCR结果显示,与1.5 Pa比较,Dvl2 mRNA的表达量在0.1 Pa FSS作用6 h和18 h时均升高(P0.05)、12 h时显著升高(P0.01);IFT88 mRNA表达量在0.1 Pa FSS作用18 h时显著升高(P0.01)。WB结果显示,与0 h比较,Dvl2蛋白的表达在0.1 Pa FSS作用18 h时升高(P0.05),在1.5 Pa FSS作用18 h时显著降低(P0.05);与1.5 Pa FSS比较,Dvl2蛋白的表达在0.1 Pa FSS作用18 h时升高(P0.05)。免疫荧光结果显示,Dvl2蛋白阳性表达随FSS作用时间的增加增多,且逐渐聚集于细胞核周边一点;IFT88蛋白阳性表达在0.1 Pa FSS作用下逐渐由细胞核向细胞质转移并聚集为一点,1.5 Pa FSS作用下逐渐减少且解聚;蛋白Dvl2、IFT88在0.1 Pa FSS作用下均定位于细胞的同一位置,在1.5 Pa FSS作用18 h内均定位于细胞的同一位置,18 h后由于蛋白IFT88发生解聚,未观察到共定位。结论层流FSS作用能够抑制PCP信号通路的转导并阻碍纤毛发生,低FSS促进其转导,且PCP信号通路可能通过Dvl2调控FSS诱导的初级纤毛发生。     

剪切应力对血管内皮细胞的影响

血管内表面覆盖有一层内皮细胞。这层内皮细胞曾被认为是血液成分与血管壁之间的屏障，仅在血管内外的养料、氧和一些大分子物质的交换时起中介作用。六十年代，病理解剖发现动脉粥样硬化常发生于血管分支处与血管弯曲部位，因而激发了人们对血管内皮细胞及其血流动力学环境的研究举。研究表明，血管内皮细胞在体内不断地受到脉动血流的剪切作用，这种血流动力学环境对内皮细胞生物学特性和内皮细胞正常功能至关重要。目前已研究发现     

Effects of shear stress on endothelial cells: go with the flow

Haemodynamic forces influence the functional properties of vascular endothelium. Endothelial cells (ECs) have a variety of receptors, which sense flow and transmit mechanical signals through mechanosensitive signalling pathways to recipient molecules that lead to phenotypic and functional changes. Arterial architecture varies greatly exhibiting bifurcations, branch points and curved regions, which are exposed to various flow patterns. Clinical studies showed that atherosclerotic plaques develop preferentially at arterial branches and curvatures, that is in the regions exposed to disturbed flow and shear stress. In the atheroprone regions, the endothelium has a proinflammatory phenotype associated with low nitric oxide production, reduced barrier function and increased proadhesive, procoagulant and proproliferative properties. Atheroresistant regions are exposed to laminar flow and high shear stress that induce prosurvival antioxidant signals and maintain the quiescent phenotype in ECs. Indeed, various flow patterns contribute to phenotypic and functional heterogeneity of arterial endothelium whose response to proatherogenic stimuli is differentiated. This may explain the preferential development of endothelial dysfunction in arterial sites with disturbed flow.     

切应力诱导间充质干细胞分化成内皮细胞的研究

目的研究绿色荧光蛋白（GFP）小鼠骨髓间充质干细胞（MSCs）体外分离培养及扩增及其在流体剪切力条件下向内皮细胞（EC）定向分化能力。方法选用GFP小鼠作为研究对象,分离培养骨髓间充质干细胞,并在体外进行细胞的扩增和传代。应用流体剪切力刺激方法对骨髓间充质干细胞进行诱导,并观测其对GFP小鼠骨髓间充质干细胞分化为内皮细胞的积极作用,对其结果进行免疫荧光检测,计算阳性细胞比例,统计分析。结果GFP小鼠骨髓间充质干细胞每扩增一代,细胞数量增加5-8倍。检测诱导后MSCs,结果显示在诱导3 d时出现vWF表达阳性细胞。结论GFP小鼠MSCs在体外的培养扩增能力强,MSCs在流体剪切应力的作用下可以转化成内皮细胞。     

Fluid shear induced endothelial cell detachment from glass - influence of adhesion time and shear stress

In this study, human umbilical vein and human saphenous vein endothelial cells were seeded on glass and exposed to fluid shear in a parallel-plate flow chamber. Cell retention, morphology and migration were studied as a function of shear stress and of adhesion time prior to exposure to shear. Three-hour and 24-h adhesion times gave rise to comparable cell retention values after 2 h of flow for both cell types. Cell retention decreased from 85 to 20% as shear stress increased from 88 to 264 dynes cm⁻² (8.8 to 26 Pa). Mean spreading areas decreased after the onset of flow, but subsequently stabilized to plateau values, which were smaller at higher shear stresses. Shape factors increased faster to higher values as cells were exposed to higher shear stresses, without any obvious preference in orientation of the cells with respect to the direction of flow. Migration was unidirectional with flow and linear with time. Migration was faster for cells which had adhered for 24 h than for cells which had adhered for 3 h and was accompanied by the presence of fibrillar structures left behind on the surface upstream of migrating cells. It is concluded that after 3 h adhesion to glass, cells have adhered with an adhesion strength that does not substantially increase during the next 21 h. However, during this time changes in cell-substratum interactions seem to occur judging by the differences in, e.g., migration rates.     

具有梯度的流体切应力促进血管内皮细胞增殖

目的探讨流体切应力(shear stress,SS)对血管内支架边缘内皮细胞(endothelial cells,ECs)增殖的影响。方法应用具有切应力梯度的平行平板流动腔(梯度切应力组)和普通矩形平行平板流动腔(恒定切应力组),分别对ECs施加0.566￣1.438Pa和1.137Pa的切应力,加载时间6h,以未施加切应力的ECs为静止对照组。流式细胞仪检测各组ECs细胞周期的变化。结果梯度切应力组ECs在受力6h后进入S期与G2+M期细胞明显多于静止对照组与稳定切应力组(P<0.05);恒定切应力组的ECs受力后进入S期与G2+M期细胞明显少于其他两组(P<0.05)。结论梯度切应力促进ECs进入分裂增殖期,而稳定的层流切应力则产生对ECs细胞周期的抑制作用。提示血管内支架植入后,继发的血流切应力改变诱导细胞进入分裂、增殖期,这可能是引起支架内再狭窄过程中血管内膜增生的原因之一。     

运动对动脉内皮功能的调控及血流剪切力的介导作用

<正>常的动脉内皮功能在调节血管通透性、维持正常的凝血功能以及免疫、炎症反应中起重要作用,受损的内皮功能则可引起血管的重构以及血栓、动脉粥样硬化等疾病的发生和发展。合理的运动训练能够通过促进一氧化氮(nitric oxide,NO)、前列环素(prostacyclin,PGI2)等物质的分泌来维持和改善动脉内皮功能,并预防动脉粥样硬化的发生。而高强度运动训练则会导致氧化应激的发生并降低NO的生物活性,从而造成内皮功能障碍及心血管疾病的发生。大量研究表明,运动引起的血流剪切力幅度及形式的变化在上述血管生物学响应中起重要的介导作用。本文综述了运动训练对动脉内皮功能的调控作用、血流剪切力的介导作用以及相关分子生物学机制等方面的研究进展,为进一步深入研究运动调控内皮功能的血流动力学机制特别是血流剪切力的重要介导作用提供参考,同时也为通过监控血流剪切力等血流动力学参数以选择最佳运动方式提供一定的科学依据。     

Shear stress and VEGF enhance endothelial differentiation of human adipose-derived stem cells

Abstract

Herein we combine chemical and mechanical stimulation to investigate the effects of vascular endothelial growth factor (VEGF) and physiological shear stress in promoting the differentiation human adipose derived stem cells (ADSCs) into endothelial cells. ADSCs were isolated and characterized; endothelial differentiation was promoted by culturing confluent cells in 50?ng/ml VEGF under physiological shear stress for up to 14 days. Afterwards, endothelial cells were seeded onto collagen or acellular aortic valve matrices and exposed to four culture conditions: shear stress + VEGF; shear stress ? VEGF; static + VEGF and static ? VEGF. After 7 days, phenotype was investigated. ADSCs subjected to shear stress and VEGF express a comprehensive range of specific endothelial markers (vWF, eNOS and FLT-1 after 7 days and CD31, FLk-1 and VE-cadherin after 14 days) and maintain the phenotype when seeded onto scaffolds. Our protocol proved to be an efficient source of endothelial-like cells for tissue engineering based on autologous ADSC.     

The influence of surface micro-structure on endothelialization under supraphysiological wall shear stress

Interaction between platelets and artificial materials within cardiovascular devices triggers blood coagulation and represents a frequent adverse response to implant deployment. Avoidance of this interaction is obtained through the generation and sustenance under flow of a confluent and stable endothelial monolayer covering the luminal device surface, altogether defined as the process of endothelialization. Supraphysiological wall shear stress (WSS) levels generated within vascular assist devices (VADs) constitute a major challenge toward endothelialization. Here we report the experimental demonstration that stable endothelialization can be achieved at supraphysiological WSS levels by pure means of appropriate surface micro-structuring. Using a custom-designed flow bioreactor we exposed endothelial monolayers to physiological and supraphysiological WSS levels and investigated the resulting integrity of cell-to-cell junctions, the cell density and the cell polarization. At physiological WSS levels, optimal endothelialization was obtained independently from surface topography. However, at higher WSS levels, only monolayers grown on appropriately micro-structured surfaces preserved optimal integrity. Under these flow conditions, endothelial cells polarized by the contact with the micro-structure and, interestingly, oriented themselves in the direction perpendicular to flow. Such endothelial layers withstood WSS levels exceeding of 100% or more the thresholds detected on flat substrates.     

激光虹膜切开术对角膜内皮细胞所受切应力影响的数值研究

目的研究激光周边虹膜切开术(laser peripheral iridoctomy,LPI)对患者眼内房水流动和角膜内皮细胞所受流体切应力的影响。方法参照文献中的人眼几何数据,在CAD软件中建立三维完整眼球几何模型,并结合有限元软件数值模拟LPI术后房水流动状态以及角膜内皮细胞所受切应力的变化,并对比LPI手术前后虹膜上不同激光开孔位置时角膜内表面切应力的数值结果。结果虹膜上激光孔的位置越靠近瞳孔中心,角膜受到的切应力越小。在激光孔离瞳孔轴距离分别为4.0、4.5、5.0、5.5 mm时,对应角膜内皮受到的最大切应力分别为16.5、25.8、57.0、179.8 m Pa。激光孔在3、6点方位时的切应力最大值比开孔在12点方位时的切应力最大值(56.95 m Pa)分别高出13.7%和4.2%。结论 LPI会改变患者眼内房水流动和角膜内皮细胞所受流体切应力。合适的开孔位置能降低角膜内皮细胞受到的切应力,减少LPI术后患者大泡角膜病变的风险。     

剪切力条件下血管内皮细胞与平滑肌细胞的相互作用

血管内皮细胞(endothelial cells,ECs)与平滑肌细胞(smooth muscle cells,SMCs)是血管壁的主要细胞,它们之间的相互作用在维持血管正常的生理功能以及心血管疾病的发生发展过程中至关重要。为真实模拟ECs与SMCs体内条件下的位置关系与生长状态,人们建立了多种共培养系统。介绍当前几种常用的能够加载流动剪切力的共培养系统,并分别比较其优势与不足;简要总结剪切力条件下ECs与SMCs相互作用对ECs与SMCs表型与分布、SMCs生长与迁移、ECs表面相关黏附分子表达的影响。研究表明,一氧化氮(NO)、细胞因子、microRNA等可以作为信号分子介导ECs与SMCs之间的相互作用。