一种简易的平行板流动腔系统的应用

为了解切应力对培养内皮细胞的生物学作用,我们建立了一种简易的平行板流动腔系统,通过本系统可以产生0∽48dyn/cm2的稳定层流切应力,应用本系统观察了切应力对内皮细胞形态、纤维状肌动蛋白(F-actin)及球状肌动蛋白(G-actin)的影响,结果发现在12h作用时间内,12、24、48dyn/cm2切应力作用下,可见细胞中央顺细胞长轴排列的应力纤维(F-actin)逐渐演变为顺血流方向排列,F-actin的含量逐渐增多,G-actin含量逐渐减少,而细胞形态无显著改变。表明这种简易平行板流动腔系统培养的内皮细胞受到的切应力作用是确实可靠的。     

切应力对与血管平滑肌细胞联合培养的内皮细胞微管骨架重构的影响

目的探讨切应力对与血管平滑肌细胞(VSMCs)联合培养的内皮细胞(ECs)中微管的聚集重构的影响,为阐明应力诱导血管重建的分子机制提供一些实验证据。方法应用ECs与VSMCs联合培养的平行平板流动腔系统,给ECs面施加15dyne/cm2的层流切应力,以静态条件下联合培养的ECs为对照组,用WesternBlot、免疫荧光细胞化学和图像分析等技术,研究切应力作用下与VSMCs联合培养的ECs的微管聚集的变化。结果静态联合培养组,ECs微管骨架的排列是稀疏、发散和无规律的。切应力诱导了ECs的微管的重构,,微管骨架变得有序,朝切应力的方向规律的排列。切应力能够促进ECs的微管聚集,与对照组相比,切应力作用下的ECs内多聚微管的数量增加,切应力作用3h,ECs内多聚微管的数量达到峰值,之后开始下降。结论切应力诱导和促进了EC的微管骨架发生重构(聚集)。结果提示:微管可能是机械应力刺激作用的靶标,应力可能通过它改变ECs的形态,影响细胞的黏附与迁移等功能。     

箭头形平行平板流室下内皮细胞形态及功能的改变

目的：观察不同血流状态下培养内皮细胞的形态及功能。 方法： 制作一种新的箭头形平行平板流室, 高速摄像微粒子示踪技术行流体分析,测量细胞形态指数和F-肌动蛋白的免疫荧光染色。 结果： 液体在入口与狭窄开始处中点的流速为249.8 mm/s，剪切应力为71.9 dyn/cm2，进入狭窄段时的流速为316 mm/s，剪切应力为88.5 dyn/cm2，细胞形态呈长纺锤形，方向沿液体流向排列，F-肌动蛋白纤维表达明显增加，呈束状，增密，上述区域液体为定常流。液体进入宽广区域后液体流速明显减慢并在两侧形成涡流甚至停滞，流速为54.4-119.6 mm/s，剪切应力为15.6-34.4 dyn/cm2，细胞近似圆形、卵圆形、多边形，F-肌动蛋白纤维表达似对照组。 结论： 新的箭头形平行平板流室是研究在不同血流状态下内皮细胞功能的良好装置。     

切应力与血管平滑肌细胞对内皮细胞增殖的影响及TGFβ1与p-Akt信号通路在其中的作用

目的探讨切应力与血管平滑肌细胞(VSMCs)对内皮细胞(ECs)增殖功能的影响及其一些分子机制。方法应用平行平板流动腔系统对单独培养的ECs以及与VSMCs联合培养的ECs施加15dyn/cm2(1dyn=10-5N)的正常切应力;Western blot技术检测反映细胞增殖能力的分子—增殖细胞核抗原(PCNA)的表达及细胞内信号转导分子Akt的磷酸化水平。静态条件下,以单独培养ECs为对照组,将ECs与VSMCs隔开培养,并应用TGFβ1封闭性抗体,观察TGFβ1在VSMCs诱导ECs增殖中的作用。结果正常切应力抑制了ECs增殖及p-Akt表达,VSMCs在与ECs联合培养及隔开培养时均明显促进ECs增殖及p-Akt表达。正常切应力部分逆转了联合培养VSMCs诱导的ECs增殖和p-Akt表达,而TGFβ1封闭性抗体能够拮抗隔开培养VSMCs诱导的ECs增殖和p-Akt表达。结论正常切应力可视为血管的保护因子,抑制ECs增殖;VSMCs通过旁分泌作用诱导了ECs增殖;TGFβ1及PI3/Akt信号分子参与了其调节过程。     

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。     

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。     

切应力对联合培养的血管内皮细胞F-肌动蛋白排列的影响

与平滑肌细胞联合培养的血管内皮细胞在静态时形态就开始发生变化,即由单独培养条件下在的多边形到联合培养条件下的长梭形。切应力作用下,联合培养的内皮细胞的形态在较短时间内发生更进一步的变化。F－actintlEylli持细胞形态及使内皮细胞与细胞外基质的粘附上起着重要的作用。为了探讨切应力对联合培养的血管内皮细胞卜肌动蛋白的排列的影响,本文应用平滑肌细胞与内皮细胞联合培养模型,将牛主动脉平滑肌细胞和内皮细胞进行联合培养,待内皮细胞形成单层后,用手行平板流动胜,将内皮细胞置于40dyn／cm’稳定层流切应力之下12、24／…     

切应力诱导内皮细胞表达IL-8基因的信号转导

目的现已确定动脉粥样硬化是一种炎症疾病,单核/巨噬细胞在动脉内膜下集聚是导致动脉粥样硬化损伤发展的基础;血流低切应力促进其损伤的发展.趋化细胞因子MCP-1对单核/巨噬细胞起强力趋化和激活作用,并有报告切应力可诱导其基因的表达.新近报道在层流作用下IL-8亦是单核/巨噬细胞与内皮细胞相互作用的重要调节因子.本研究旨在观察切应力诱导人脐静脉血管内皮细胞IL-8mRNA的表达,探讨Toll/NF-κB信号转导通路对其基因激活的介导作用.方法4.2dyn/cm2层流切应力处理人脐静脉血管内皮细胞,提取总RNA,应用RT-PCR和Northern杂交技术检测切应力作用不同时间后IL-8、TLR-2和TLR-4mRNA的表达.免疫印迹技术检测IκB磷酸化和降解.免疫荧光细胞化学染色检测NF-κB胞核易位.结果切应力作用0.5h后IL-8mRNA表达逐渐增高,2h时达到很高水平.胞浆蛋白IκB和磷酸化IκB的免疫印迹显示4.2dyn/cm2层流切应力作用10min后磷酸化IκB水平即显著增加,30min后又逐渐下降,与之相应IκB含量随切应力作用时间而逐渐下降,到1h时几乎测不出,表明在切应力作用下内皮细胞胞浆NF-κB抑制因子IκB发生了磷酸化和降解.NF-κBp65亚基免疫细胞化学染色显示在4.2dyn/cm2切应力作用0.5h后内皮细胞核逐渐着色,1.5h时细胞核几乎全着色,表明胞浆NF-κB在切应力作用下发生了易位,从胞浆进入胞核.RT-PCR检测和Northern杂交分析显示内皮细胞固有表达TLR-2和TLR-4mRNA,在4.2dyn/cm2切应力作用1h后TLR-4mRNA的表达显著增强,而TLR-2mRNA的表达变化不明显.结论流体切应力可诱导血管内皮细胞表达IL-8,活化转录因子NF-κB.在切应力作用下内皮细胞TLR-4mRNA表达增强,提示Toll/NF-κB信号转导通路可能参与动脉粥样硬化性炎症反应.     

切应力对与内皮细胞联合培养的血管平滑肌细胞粘附的影响及其机制

目的　细胞粘附是细胞迁移的关键性起始步骤。研究切应力和内皮细胞(endothelial cells, ECs)对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)粘附的影响及其可能的信号通路，为探讨流体切应力诱导的血管壁细胞迁移行为的机制提供一些实验依据。方法　应用VSMCs和ECs联合培养流动腔系统，对ECs面施加1.5 Pa切应力，12 h；以静止状态下，单独培养的VSMCs以及与ECs联合培养的VSMCs 为对照，应用细胞粘附实验和Western Blot技术，观察切应力对与EC联合培养的VSMCs粘附的影响及蛋白激酶B(PKB/Akt)磷酸化水平表达变化。结果　静态联合培养12 h，VSMCs的粘附能力明显增强，同时磷酸化Akt的表达平行增高。切应力作用下，明显抑制了联合培养的VSMCs粘附，同时磷酸化Akt的表达平行降低。结论　生理大小切应力明显抑制了ECs诱导的VSMCs粘附，其中Akt信号通路起了关键作用。     

不同加载时间流体剪切力对成骨细胞中基质金属蛋白酶-1和基质金属蛋白酶抑制剂-1表达的影响

目的:研究加载不同时间流体剪切力对成骨细胞基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶抑制因子(TIMP-1)表达的影响。方法:利用自行设计的平行平板流体剪切力加载装置,对MC3T3-E1成骨细胞施加12 dyn/cm2流体剪切力0、15、30、45、60 min,采用蛋白免疫印记实验检测MMP-1和其抑制剂TIMP-1的表达水平。结果:对体外培养的MC3T3-E1细胞加载12 dyn/cm2流体剪切力,随着加载时间的延长,MMP-1表达上调,TIMP-1表达水平下调,在45 min左右达到高峰。结论:加载不同时间的12 dyn/cm2流体剪切力能够上调MC3T3-E1成骨细胞MMP-I的表达,下调TIMP-1的表达,加载45 min最合适。     

用于内皮细胞与平滑肌细胞联合培养的流动腔系统

内皮细胞与平滑肌细胞的联合培养是体外研究这两种细胞生物学特性的重要手段。静态条件下联合培养的内皮细胞的形态及功能特性与在体条件下均有差异,这可能是没有血流切应力对内皮细胞直接作用的结果。为了了解切应力对与平滑肌细胞联合培养的内皮细胞的生物学作用,本文建立了用于多孔ＰＥＴ膜联合培养模型的力学系统,通过本系统可以产生０～４０ｄｙｎ／ｃｍ２的稳定层流切应力,并应用本系统观察了切应力对联合培养的内皮细胞的形态学影响,结果表明,在４０ｄｙｎ／ｃｍ２切应力作用下,与平滑肌细胞联合培养的内皮细胞沿流体方向发生了重排。这一系统的建立为研究切应力作用下与平滑肌细胞联合培养的内皮细胞的形态和功能提供了手段。     

Steady unidirectional laminar flow inhibits monolayer formation by human and rat microvascular endothelial cells.

Endothelialization of artificial vascular grafts is rapid and complete in numerous animal models, including dogs and rats, but not in human patients. One possible explanation for this well-known, yet puzzling observation might be that monolayer formation of human endothelial cells (ECs), and of canine or rodent ECs, is affected differently by flow-induced shear stress. To begin testing this hypothesis, the authors wounded confluent monolayers of cultured rat and human ECs and exposed these cultures for 20 h to unidirectional steady laminar shear stress of 10 dyn/cm(2) induced by fluid flow perpendicular to the wound boundaries. In comparison to experimental control cultures simultaneously maintained under static (no-flow) conditions, flow-induced shear stress attenuated the monolayer formation (sheet migration) in both human and rat ECs. In brief, compared to control, the average human EC monolayer formation under shear was reduced by 33% whereas the average rat EC monolayer formation was reduced by 34%. Furthermore, the cell responses showed a dependence on fluid flow direction that differed per species. When exposed to shear stress, human EC monolayer formation was reduced by 16% in the upstream direction (opposing the direction of flow) and reduced by 50% in the downstream direction (with the direction of flow), whereas rat EC monolayer formation was reduced by 64% upstream and showed no change downstream. These findings suggest that although overall monolayer formation is inhibited by fluid-induced shear stress to the same extent in both species, there are cell type- and/or species-dependent migration responses to fluid-induced shear stress, and that different flow conditions possibly contribute to species-specific patterns of endothelialization.     

p38 Mitogen-activated protein kinase activation in endothelial cell is implicated in cell alignment and elongation induced by fluid shear stress.

Fluid shear stress is thought to be important in maintaining the phenotype of endothelial cells (ECs) in vivo. The purpose of the study was to determine the effect of varying levels of laminar shear stress on EC elongation and alignment and the role of p38 mitogen-activated protein kinase (MAPK) on the morphologic change induced by shear stress. Cultured bovine aortic ECs were subjected to 1, 4, 7, 14, or 20 dyne/cm(2) laminar steady shear stress. On morphometric analysis of static ECs, the average orientation angle was 41 degrees , whereas after 24 h shear stress at 1, 4, 7, 14, and 20 dyne/cm(2) the angles were 34 degrees, 33 degrees, 16 degrees, 11 degrees, and 10 degrees, respectively. The shape index of static ECs was 0.76, whereas the indexes of ECs exposed to shear stress were 0.72, 0.72, 0.65, 0.50, and 0.47, respectively. The time and the magnitude of activation of p38 MAPK were dependent on the level of shear stress. The results indicate that a minimum shear stress of 7 to 14 dynes/cm(2) is necessary for cell alignment and elongation and this correlates with activity of p38 MAPK. ECs exposed to shear stress in the presence of the p38 MAPK inhibitor SB-203580 did not orient in any manner and the shape index was similar to the static cells.     

The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(L-lactide-co-epsilon-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm(2). The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm(2)) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm(2)) to a high shear stress (19.6 dyn/cm(2)) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.     

Gene response in endothelial cells cultured on engineered surfaces is regulated by shear stress

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.     

Micro Sensors: Linking Real-Time Oscillatory Shear Stress with Vascular Inflammatory Responses

The important interplay between blood circulation and vascular cell behavior warrants the development of highly sensitive but small sensing systems. The emerging micro electro mechanical systems (MEMS) technology, thus, provides the high spatiotemporal resolution to link biomechanical forces on the microscale with large-scale physiology. We fabricated MEMS sensors, comparable to the endothelial cells (ECs) in size, to link real-time shear stress with monocyte/EC interactions in an oscillatory flow environment, simulating the moving and unsteady separation point at arterial bifurcations. In response to oscillatory shear stress (tau) at +/- 2.6 dyn/cm2, time-averaged shear stress (tauave) = 0 at 0.5 Hz, individual monocytes displayed unique to-and-fro trajectories, undergoing rolling, binding, and dissociation with other monocyte, followed by solid adhesion on EC. Incorporating with cell-tracking velocimetry, we visualized that these real-time events occurred over a dynamic range of oscillating shear stress between +/- 2.6 dyn/cm2 and Reynolds number between 0 and 22.2 in the presence of activated adhesion molecule and chemokine mRNA expression.     

Endothelial barrier function under laminar fluid shear stress

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress-induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 ohms cm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of alpha-, beta-, and gamma-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress-induced junction rearrangement. Additionally, catenins were partly colocalized with beta-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress-dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.