扰动剪切流场对体外培养的血管内皮细胞LDL受体的影响

利用能更好体外模拟血液流动情况的带后向台阶的流动腔装置,对在扰动剪切流场作用下血管内皮细胞低密度脂蛋白(LDL)受体的表达进行了研究.结果显示,扰动流作用于内皮细胞后,细胞表面LDL受体表达较层流区域降低.提示扰动剪切流场会降低LDL受体的表达而降低对LDL的转运,引起LDL在内皮下沉积,从而易于诱发动脉粥样硬化.     

用复合液滴模型研究稳定剪切流动下黏附于血管表面白细胞的变形

变形作用是黏附于血管表面的白细胞响应外界流体作用的重要特征，然而，尚不清楚白细胞的细胞核是否伴随细胞而发生变形。我们用复合液滴模拟黏附于血管表面的白细胞，根据二维计算流体动力学方法研究稳定剪切流动下白细咆及其细咆核发生变形的生物力学机制。结果表明：(1)外界流场雷诺数是引起细咆变形的重要原因。随着雷诺数的增大，细胞变形也增大；(2)细胞核随细胞的变形而发生了变形，但细胞的变形指数总是大于其细胞核，表明细胞核更能耐受剪切流动；(3)当雷诺数增大到一定值时，细胞和细胞核都不能进一步变形，变形指数达到峰值；(4)压力分布曲线表明，在细胞的下游形成一个高压区，提供促使细胞受力达到平衡的升力，从而阻止了细胞的进一步变形：因此，具有高粘性的细胞核在细胞的变形过程中发挥特殊作用。     

黏附于血管壁表面的白细胞在稳定剪切流动下发生变形的生物力学模型

白细胞与血管表面的黏附是重要的生物医学工程问题，引起了学者们的广泛研究。我们用复合液滴来模拟黏附于血管表面的白细胞，根据二维计算流体动力方法研究了流体切应力作用下白细胞黏附引起的压力分布。同时，通过引入“变形指数”的概念，研究稳定剪切流动下白细胞变形的生物力学特性，数值结果表明：（1）随着初始接触角，毛细血管数，外界流场雷诺数的增大，细胞的变形也增大，而细胞浆比细胞核更易于变形，表明细胞核更能耐受切流动；（2）当切应力增大到一定值时，细胞不能进一步变形，变形指数达到峰值；（3）压力分布曲线表明，在细胞的下游形成一个高压区，提供促使细胞受力达到平衡的升力，从而阻止了细胞的进一步变形，我们关于细胞核变形的结果有助于理解白细胞如何将外界流体作用力（如切应力）等力学信号向核内转导的生物力学机理。     

二维脉动流场中内皮细胞表面切应力分布的数值模拟

本文对三种生理相关的正弦入口速度波形的脉动流场内,VEC表面的切应力分布进行了有限差分方法的数值模拟.结果表明(1)脉动流场与稳态流场中内皮细胞表面的流场分布完全不同,脉动流场中内皮细胞表面的切应力变化幅值远大于稳态流场中的值.(2)同一时刻,每个VEC上的切应力分布是不均匀的,细胞形状影响其表面的切应力分布.(3)在脉动周期的不同时刻,细胞表面的切应力分布也是不均匀的,切应力分布随时间的变化波形与入口波形相类似,但相位有所超前.(4)细胞的伸长主要取决于EC表面的最大平均切应力大小.(5)本章的计算结果可以用于对Helmilinger实验现象的解释.     

二维脉动流场中内皮细胞表面切应力分布的数值模拟

本文对三种生理相关的正弦入口速度波形的脉动流场内,VEC表面的切应力分布进行了有限差分方法的数值模拟。结果表明：（1）脉动流场与稳态流场中内皮细胞表面的流场分布完全不同,脉动流场中内皮细胞表面的切应力变化幅度远大于稳态流场中的值。（2）同一时刻,每个VEC上的切应力分布是不均匀的,细胞形状影响其表面的切应力分布。（3）在脉动周期的不同时刻,细胞表面的切应力分布也是不均匀的,切应力分布随时间的变化波形与入口波形相类似,但相位有所超前,（4）细胞的伸长主要取决于EC表面的最大平均应力大小。（5）本章的计算结果可以用于Helmilinger实验现象的解释。     

稳定黏附的白细胞与血液流动的耦合作用

提出了血液流动与牢固黏附的白细胞之间发生耦合作用的理论模型,根据计算流体动力学方法推导血流作用下白细胞的变形指数、表面应力、压力分布。在此基础上,用激光多谱勒测速法对上述模型进行实验研究。结果表明,白细胞的变形随初始接触角、血流雷诺数的增大而增大;同时,血流引起细胞表面的应力和压力重新分配,但最大剪应力总是位于细胞表面的顶点。由此推测,细胞表面应力分布的不均一可能对细胞的形态与功能变化有重要影响。     

流场压力信号变化对内皮细胞形态和黏附功能的影响

人体血循环系统是一个有压的封闭系统,血管流场中,由于血管自然走行或病变导致其几何形状的改变,必然导致局部流场压力发生变化,进而可能引起血管内皮细胞发生一系列生理或病理变化.本研究拟考察流场内培养的内皮细胞(Endothelial cells, ECs)在不同压力信号作用下细胞浆内肌动蛋白纤维F-actin排列、表达强度以及血管内皮细胞黏附分子(Vascular cell adhesion molecule, VCAM)和整合素Integrin αVβ3等细胞黏附分子的表达强度,探讨分析其与流场内压力变化之间的关系.通过设置静态培养、单纯剪切力对照组,应用免疫荧光双重标记、激光共聚焦扫描显微镜和计算机图像分析等技术,观察了在平板流室内于不同压力加载下培养的内皮细胞表面VCAM和Integrin αVβ3 的表达强度及细胞骨架F-actin 的变化.研究结果初步表明,随着压力降值的变化,内皮细胞表面VCAM和Integrin αVβ3以及F-actin的表达强度呈现出明显的规律性改变现象.压力降信号的改变导致了内皮细胞骨架F-actin的变化,同时影响了以VCAM为代表的细胞黏附功能和以Integrin αVβ3为代表的压力信号跨膜传递通路的生理功能.     

用激光测速法研究稳定黏附的白细胞与血液流动的相互作用

提出了血液流动与牢固黏附的白细胞之间相互作用的理论模型,根据计算流体动力学方法推导血流作用下白细胞表面应力、压力分布,以及由于黏附的白细胞对血流阻力的影响.在此基础上,用激光测速法对上述模型进行实验研究.结果表明,血流阻力、压力梯度随血流雷诺数、白细胞直径的增大而增大;细胞表面的应力重新分布,但最大剪应力总是位于细胞表面的顶点.我们认为细胞表面应力分布的不均一可能对白细胞的形态与功能变化有重要作用.     

稳定层流剪应力对内皮细胞骨架调节蛋白VASP表达的影响

为探讨生理强度的稳定层流剪应力对内皮细胞骨架actin相关蛋白VASP特征影响规律，我们采用胰蛋白酶消化法分离人脐静脉内皮细胞(HUVECs)，模拟体内流动环境，建立平行板流动腔模型。利用细胞图像分析系统和ALEXA488—若丹明一次毒蕈环肽双标记法，观察内皮细胞在稳态层流下形态、actin排列变化与VASP分布变化之间的规律。采用Western blot定量动态检测细胞内VASP表达及磷酸化的水平。结果表明，内皮细胞在10dyn／cm^2剪切作用后，随时间细胞逐渐延长，长轴趋于剪应力作用方向排列，细胞与静息态的细胞相比，细胞内骨架沿剪应力方向重组，与此同时VASP表达增强，沿着actin纤维呈点状分布，尤其集中在细胞膜下actin末端区域；Western blot检测显示在剪切后，细胞内VASP出现快速磷酸化，VASP总体表达量增加，2h达高峰后逐渐恢复，8h后再次逐渐升高。以上结果提示血流动力学特性中剪应力引起了细胞胞质内骨架蛋白分子重组，血管内皮细胞形态改变，在此过程中，VASP发挥骨架调节蛋白的作用。     

038 不同剪切应力对血管内皮细胞ICAM-1分布的影响

目的: 剪切应力是血液在血管内流动时对内皮细胞产生的一种机械刺激, 一定程度的剪切应力对于维持血管的正常结构和功能十分重要. ICAM-1是血管内皮细胞等产生的一类免疫球蛋白超家族粘附分子, 除介导免疫、炎症反应及细胞间粘附和连接外, 还能传递多种细胞信号, 引起细胞内骨架蛋白结构与功能的改变. 故此, 研究在不同剪切应力的作用下, 血管内皮细胞表面粘附分子ICAM-1分布的改变及其机制. 方法: 将ECV304细胞常规培养在长方形的玻片上, 然后分别置于剪切应力为0.2 Pa、 1 Pa(相当于正常状况下动脉血管内的剪切应力)、 2 Pa的流场中, 温度37 ℃. 4 h后取出玻片, 先用鼠抗人ICAM-1单克隆抗体(1∶20)孵育20 min, PBS冲洗, 再用标记有AlexaR荧光的羊抗鼠IgG(1∶50)作为二抗标记, 1%多聚甲醛和0.5% Triton-X100渗透和固定后, 在Olympus IX70荧光显微镜下观察ICAM-1的分布状况. 结果: 在正常状态下(1 Pa), ICAM-1主要分布在细胞间连接处, 而在较低剪切应力(0.2 Pa)和较高剪切应力(2 Pa)作用下, ICAM-1则呈现出细胞表面散在分布的特征. 结论: 过低或过高的剪切应力都会引起血管内皮细胞表面ICAM-1分布的改变.     

Oxygen tension modulates Ca2+ response to flow stimulus in endothelial cells exposed to hydrostatic pressure.

The effects of oxygen gas tensions and hydrostatic pressure on intracellular calcium, [Ca2+]i, response to a flow stimulus in endothelial cells were investigated. Cultured bovine aortic endothelial cells (BAECs) were exposed to a hydrostatic pressure of 100 mmHg under low oxygen gas tensions and were subsequently subjected to a 1 minute mechanical stimulation of fluid shear stress of 20 dynes/cm2. The [Ca2+]i response in BAECs was measured using a fluorescent indicator, Calcium Green-1. The maximum intensity for low oxygen tension was significantly lower than that for normal oxygen tension, which provides evidence that low oxygen tension regulates cellular functions downward. Moreover, preloading of hydrostatic pressure also reduced the increases in [Ca2+]i. These results suggest that BAECs in venous system, where oxygen tension and hydrostatic pressure are lower than those in arterial system, may be less sensitive to fluid flow. A separate observation showed that low oxygen tension did not significantly affect the cell morphology. In contrast, BAECs exposed to hydrostatic pressure showed marked elongation with no predominant orientation and the F-actin filament distributions were rearranged, indicating centrally located thick stress fibers. For better understanding of endothelial cell physiology, it is very important to elucidate the effect of oxygen gas tensions together with mechanical environment on endothelial cell responses.     

流动切应力对培养的血管内皮细胞形态的影响

为探索培养的血客内皮细胞承受流体冲击的能力，以便在人工心脏瓣膜表面形成一个抵抗血流切应力作用强的血管内皮细胞层，在我们研制的内皮细胞切应力反应测试装置上，观察了培养在盖玻片表现的牛主动脉内皮细胞形成单层细胞后在切应力分别在１５ｄｆｙｍｅｓ／ｃｍ＾２２４小时和１１５ｄｙｍｓ／ｃｍ＾２８小时作用下细胞的形态张脱落情况。     

Mechanotransduction of bone cells<Emphasis Type="Italic">in vitro</Emphasis>: Mechanobiology of bone tissue

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E₂ values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E₂. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.     

不同条件培养的内皮细胞耐受流体剪切力的比较研究

为提高内皮细胞（EC）与移植间的粘附,研究其可能的机制,我们采用改进的流室装置比较流动培养的EC与静态培养的EC对剪切力的耐受强度,预铺纤维粘连蛋白,层粘连蛋白对其对其粘附的影响,并研究2了EC骨架成分肌动蛋白丝的分布及在剪切力作用下的变化,结果显示流动培养组的细胞残留明显多于相应静态培养组,预辅纤维连接蛋白可显著提高静态培养EC的残留,加入纤维连接蛋白和层粘连蛋白效应更明显,而预铺的基质对流动培养组结果影响较小,剪切力作用下EC的肌动蛋白丝有序排列,并形成张力纤维,提示流动培养可显著提高EC对剪切力的耐受,其机制与剪切力诱导肌动蛋白丝重建,张力纤维形成,细胞形态的变化有关。     

Cellular Fluid Mechanics and Mechanotransduction

Mechanotransduction, the transformation of an applied mechanical force into a cellular biomolecular response, is briefly reviewed focusing on fluid shear stress and endothelial cells. Particular emphasis is placed on recent studies of the surface proteoglycan layer (glycocalyx) as a primary sensor of fluid shear stress that can transmit force to apical structures such as the plasma membrane or the actin cortical web where transduction can take place or to more remote regions of the cell such as intercellular junctions and basal adhesion plaques where transduction can also occur. All of these possibilities are reviewed from an integrated perspective.     

Computational modelling of the mechanical environment of osteogenesis within a polylactic acid–calcium phosphate glass scaffold

A computational model based on finite element method (FEM) and computational fluid dynamics (CFD) is developed to analyse the mechanical stimuli in a composite scaffold made of polylactic acid (PLA) matrix with calcium phosphate glass (Glass) particles. Different bioreactor loading conditions were simulated within the scaffold. In vitro perfusion conditions were reproduced in the model. Dynamic compression was also reproduced in an uncoupled fluid-structure scheme: deformation level was studied analyzing the mechanical response of scaffold alone under static compression while strain rate was studied considering the fluid flow induced by compression through fixed scaffold. Results of the model show that during perfusion test an inlet velocity of 25 μm/s generates on scaffold surface a fluid flow shear stress which may stimulate osteogenesis. Dynamic compression of 5% applied on the PLA–Glass scaffold with a strain rate of 0.005 s^?1 has the benefit to generate mechanical stimuli based on both solid shear strain and fluid flow shear stress on large scaffold surface area. Values of perfusion inlet velocity or compression strain rate one order of magnitude lower may promote cell proliferation while values one order of magnitude higher may be detrimental for cells. FEM–CFD scaffold models may help to determine loading conditions promoting bone formation and to interpret experimental results from a mechanical point of view.     

A device for subjecting vascular endothelial cells to both fluid shear stress and circumferential cyclic stretch

The proposal of the role of mechanical forces as a localizing factor of atherosclerosis has led many researchers to investigate their effects on vascular endothelial cells. Most previous efforts have concentrated on either the fluid shear stress, which results from the flow of blood, or the circumferential “hoop” stretch, which results from the expansion of the artery during the cardiac cycle. In fact, arterial endothelial cells are subjected to both fluid shear stress and cyclic hoop stretchin vivo. Therefore, a more complete investigation of mechanical phenomena on endothelial cell behavior should include both kinds of mechanical stimuli. This study was undertaken to design an experimental apparatus that could subject cultured vascular endothelial cells to simultaneous physiologic levels of both shear stress and cyclic hoop stretch. The experimental apparatus consists of four cylindrical elastic tubes so that the following conditions may be studied: (a) static conditions; (b) shear stress only; (c) hoop stretch only; and (d) shear stress and hoop stretch. In order to establish the functional capabilities of the apparatus, bovine pulmonary artery endothelial cells were cultured in the tubes, and their morphology and f-actin structur were observed with confocal microscopy. The cells remained healthy and attached to the walls throughout the 24 hr experiment. Preliminary results indicated that the alignment of endothelial cells subjected to shear stress was significantly enhanced by the addition of hoop strain.     

Finite-Element Stress Analysis of a Multicomponent Model of Sheared and Focally-Adhered Endothelial Cells

Hemodynamic forces applied at the apical surface of vascular endothelial cells may be redistributed to and amplified at remote intracellular organelles and protein complexes where they are transduced to biochemical signals. In this study we sought to quantify the effects of cellular material inhomogeneities and discrete attachment points on intracellular stresses resulting from physiological fluid flow. Steady-state shear- and magnetic bead-induced stress, strain, and displacement distributions were determined from finite-element stress analysis of a cell-specific, multicomponent elastic continuum model developed from multimodal fluorescence images of confluent endothelial cell (EC) monolayers and their nuclei. Focal adhesion locations and areas were determined from quantitative total internal reflection fluorescence microscopy and verified using green fluorescence protein–focal adhesion kinase (GFP–FAK). The model predicts that shear stress induces small heterogeneous deformations of the endothelial cell cytoplasm on the order of <100 nm. However, strain and stress were amplified 10–100-fold over apical values in and around the high-modulus nucleus and near focal adhesions (FAs) and stress distributions depended on flow direction. The presence of a 0.4 μm glycocalyx was predicted to increase intracellular stresses by ∼2-fold. The model of magnetic bead twisting rheometry also predicted heterogeneous stress, strain, and displacement fields resulting from material heterogeneities and FAs. Thus, large differences in moduli between the nucleus and cytoplasm and the juxtaposition of constrained regions (e.g. FAs) and unattached regions provide two mechanisms of stress amplification in sheared endothelial cells. Such phenomena may play a role in subcellular localization of early mechanotransduction events.