周期性张应变调控血管平滑肌细胞黏附血小板微体及其在自噬中的作用

目的探讨周期性张应变力学刺激对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)与血小板微体(platelet-derived microparticles,PMPs)黏附能力的影响,以及黏附的PMPs对VSMCs自噬的调控作用。方法应用FX-5000T张应变加载系统,对体外培养VSMCs施加5%幅度的生理性张应变和15%幅度的高张应变;应用流式细胞术检测不同张应变作用的VSMCs与PMPs的黏附;免疫荧光检测PMPs刺激24 h后自噬标志分子微管相关蛋白轻链3(autophagy microtubule associated protein light chain 3,LC3)的表达水平; Western blotting检测PMPs刺激24 h后VSMCs自噬相关蛋白(autophagy related protein,Atg)的表达水平。结果与5%生理性张应变加载相比,15%高张应变加载24 h能显著增强VSMCs与PMPs的黏附水平,提示高张应变促进PMPs与VSMCs的黏附。免疫荧光和Western blotting结果显示,PMPs刺激可显著上升VSMCs中自噬标志蛋白LC3表达,同时Western blotting检测到PMPs刺激后Atg5、Atg7、Atg12蛋白表达水平显著上升。结论高张应变可以促进VSMCs黏附PMPs,黏附的PMPs可能通过增加Atg5、Atg7、Atg12、LC3表达,从而增强VSMCs自噬。     

不同频率张应变对大鼠血管平滑肌细胞α-肌动蛋白表达的影响

目的研究不同频率张应变对血管平滑肌细胞表型转换的影响。方法应用FX-4000T细胞应变加载系统,对体外培养的大鼠血管平滑肌细胞施加10％应变,频率分别为0．5Hz、1Hz和2Hz,加载时间12h和24h后,采用Real time RT-PCR、Western blot、免疫荧光化学等技术检测不同频率张应变下血管平滑肌细胞的alpha-肌动蛋白（α-actin）的表达变化。以未加载张应变的血管平滑肌细胞为对照组。结果血管平滑肌细胞的α-actin的蛋白和RNA表达量在1Hz条件下比相同时相点的其他频率组均高。结论 不同频率的张应变可以影响血管平滑肌细胞α-actin的表达量,提示应变频率的改变可能参与血管平滑肌细胞表型转换的调节。     

不同频率张应变对大鼠血管平滑肌细胞外基质的影响

为研究不同频率张应变对血管平滑肌细胞(VSMC)细胞外基质(ECM)的影响,探讨不同频率张应变与血管重建(remodeling)的关系,本文应用 FX-4000TTM细胞应变加载系统,对体外培养的大鼠VSMC施加10%张应变,频率分别为0.5、1和2 Hz,加载时间为24 h,以未加载张应变的VSMC为对照组.采用Real-time RT-PCR、western blot等技术检测不同频率张应变对fibronectin、collagen I和collagen III表达的影响,以及可能参与调节的蛋白激酶p38的活性变化.结果显示:①张应变频率可以明显影响细胞外基质Fibronectin、collagen I和collagen III的mRNA的表达,其影响效果与频率大小是一种非线性关系;②蛋白激酶p38参与调节了一定频率张应变诱导的ECM的表达变化.结果表明不同频率的张应变可以影响VSMC细胞外基质表达的变化,提示频率的改变可能参与调节细胞外基质的合成与分泌;在应力引起的血管重建中频率的改变可能起着重要的作用.     

机械张应变诱导蛋白激酶B活化对血管平滑肌细胞迁移的影响

目的研究机械张应变诱导蛋白激酶B活化对大鼠血管平滑肌细胞迁移能力的影响。方法应用FX-4000T细胞应变加载系统,对大鼠血管平滑肌细胞施加牵拉幅度为15%、频率为1Hz的张应变。以Transwell和Westernblot等方法观察张应变作用下蛋白激酶B磷酸化和血管平滑肌细胞迁移能力的变化,以未加载张应变的血管平滑肌细胞为对照组。结果与对照组相比,机械张应变增加细胞中蛋白激酶B磷酸化水平,促进血管平滑肌细胞的迁移;PI3K的特异性抑制剂Wortmannin抑制张应变诱导的蛋白激酶B的磷酸化,降低了血管平滑肌细胞迁移能力。结论机械张应变通过上调蛋白激酶B磷酸化水平促进了血管平滑肌细胞迁移,提示蛋白激酶B信号通路参与机械张应变条件下血管平滑肌细胞迁移过程的信号传导。     

磁共振扩散高阶张量成像的脑白质纤维微结构模型及特征提取算法

在基于扩散加权磁共振成像(DW-MRI)的脑白质纤维微结构建模中,高阶张量成像模型(HOT)是解决常用二阶张量模(DTI)难以刻画复杂纤维结构问题的新方法,但是存在纤维特征方向提取复杂、计算量大等问题。本研究基于任意阶次的正定高阶张量理论,提出一种能够快速获得任意阶张量纤维特征方向的迭代搜索算法。该方法根据张量模型的特征,首先利用网格细分快速确定特征方向的大致区域,进一步针对该区域进行细分,从而迭代获得精确的高阶张量模型特征方向。该方法解决了现有符号计算方法易于陷入局部极值点或者搜索不收敛带来的误差和计算效率问题。为了测试所提出算法的有效性,首先利用仿真数据分析了算法对不同数目、不同交角纤维的识别能力和特征方向的计算精度;同时利用实际DW-MRI临床数据进行了测试。结果表明,相比于现有的符号计算方法,所提出的算法可以在6阶以及更高阶模型下获得稳定的纤维特征方向。     

力学刺激对巨噬细胞极性的影响

目的 探讨力学刺激对巨噬细胞极性的影响。方法 使用不同幅度、时间张应变刺激RAW264.7细胞,CCK-8试剂检测细胞活性以明确使用的力学刺激参数。将RAW264.7细胞诱导为M1型巨噬细胞,并施加10%幅度、2 Hz张应变。使用CCK-8、流式细胞仪检测张应变对细胞活性、凋亡的影响。运用实时荧光定量聚合酶链反应仪（quantitative real-time polymerase chain reaction, qRT-PCR）检测张应变对M1型巨噬细胞炎症相关基因表达量的影响。结果 在刺激3 h后,15%和20%幅度、2 Hz张应变会显著抑制细胞活性（P＜0.05）,而10%幅度、2 Hz张应变不会对RAW264.7活性产生抑制作用（P＞0.05）。10%幅度、2 Hz张应变对M1型巨噬细胞凋亡、活性无显著影响（P＞0.05）。10%幅度、2 Hz张应变可以显著抑制M1型巨噬细胞炎症相关因子白细胞介素-1β（interleukin-1β,IL-1β）、肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）的表达（P＜0.05）。结论 10%幅度、2 Hz力学刺激可抑制M1型巨噬细胞,促使其向M2型转化。力学刺激可能成为炎症相关疾病的一种治疗手段。     

TNF-α促进内皮源性微体的数量与ICAM-1表达

目的探讨高张应变调控的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对内皮源性微体(endothelial microparticles,EMPs)数量与表面细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)表达的作用。方法采用Flexercell细胞张应变加载系统对大鼠胸主动脉内皮细胞(endothelial cells,ECs)分别施加5%(模拟正常生理状态)和18%(模拟高血压状态)幅度的周期性张应变,加载频率均为1.25 Hz,加载持续时间为24 h,实时PCR检测不同幅度张应变条件下ECs的TNF-αmRNA表达水平。之后应用TNF-α刺激大鼠胸主动脉ECs,收集上清液,超速离心提取得到内皮源性微体(endothelial microparticles,EMPs);用亲脂性苯乙烯基(lipophilic styryl)以及透射电镜对EMPs进行形态鉴定;流式细胞术对TNF-α刺激产生的Annexin V阳性EMPs进行计数,并检测EMPs表面ICAM-1的表达。结果与5%正常张应变组相比,18%高张应变条件下ECs的TNF-α表达水平显著上升。TNF-α能够显著上调ECs产生Annexin V阳性的EMPs数量,且TNF-α刺激ECs产生的EMPs表面ICAM-1表达量显著增加。结论高张应变条件下ECs高表达TNF-α可能介导了EMPs产生和表面ICAM-1高表达。研究结果为后续探讨EMPs在血管重建力学生物学机制中的作用提供新的实验证据。     

生理性周期性张应变通过激活AMPK磷酸化抑制血管平滑肌细胞迁移

目的 探讨细胞能量代谢的关键调节因子 AMP激活的蛋白激酶AMPK在血管平滑肌细胞（vascular smooth muscle cells, VSMCs）响应生理性周期性张应变力学刺激后对VSMCs迁移的影响。方法 采用 Flexcell-5000T体外细胞张应变加载系统,对大鼠原代培养的 VSMCs 施加10%幅度、1.25 Hz 频率的周期性张应变,模拟VSMCs在体内的生理性力学环境;以未加载周期性张应变的静态细胞为对照组,Western blotting 检测 VSMCs的 p-AMPK蛋白表达;划痕实验检测 VSMCs 迁移功能。结果 与静态组的细胞相比,生理性周期性张应变加载24 h后显著减少划痕愈合面积,提示生理性周期性张应变抑制VSMCs迁移;生理性周期性张应变加载3 h后,VSMCs的p-AMPK蛋白表达显著升高,而加载24 h后p-AMPK蛋白表达显著降低。在生理性周期性张应变加载条件下,孵育AMPK抑制剂可以在张应变加载3 h后显著降低 p-AMPK蛋白表达,而在张应变加载24 h后显著促进VSMCs迁移;在静态条件下孵育AMPK激活剂 AICAR 3 h后显著诱导p-AMPK蛋白表达,孵育24 h后显著抑制VSMCs迁移;提示p-AMPK蛋白表达参与调控VSMCs迁移。结论 生理性周期性张应变能通过激活p-AMPK蛋白表达,进而抑制VSMCs迁移,提示生理性周期性张应变调控VSMCs迁移对维持血管稳态具有重要意义。     

扩散张量MR成像研究

本文一方面综述了8年来国际上扩散张量磁共振成像(DTI)的研究进展情况。包括总结了到目前为止所使用的4种提高DTI成像精度的方法，并指出精度的提高依赖于成像中脉冲序列的优化、实验方法的完善和后处理算法两个方面。文中比较了目前研究扩散张量的两种基本模型：扩散张量模型和q-空间模型。指出这两种模型侧重应用于不同的场合。前者侧重于研究器官或宏观组织中的扩散，而后者侧重于研究小到细胞尺度(μm量级)的扩散行为。两者在应用研究方面是互补的，所要求的实验条件是有差别的。另一方面结合文献对扩散张量模型的实验条件进行了理论分析。认为b因子取得过高并不合理。并用DTI实验数据和结果进行了初步验证。为了改进扩散张量模型本文探讨了考虑多指数衰减的设想。文中综述了DTI的导出量和一些实验结果，在此基础上分析了设计和优化各向异性指标(DAI)的原则。对于DTI的重要导出结果神经纤维柬成像(fiber tractography)重点分析了造成其成像精度不高的主要因素，指出了改进纤维束传导方向甄选算法和寻求纤维束方向场的几何性质是两种可能的解决办法。     

一种血管张应力体外加载装置的实验研究

目的研制一种血管张应力体外加载装置,研究弹性基底(硅胶片)上的张应力、张应变分布。方法基于基底形变加载技术,研制一种接近人体血液动力学环境的血管张应力体外加载装置。利用装置中的摄像机拍摄硅胶片拉伸前后硅胶片网格点的图像并转化为数字图像,使用Matlab软件对网格点的位置特征进行计算,从而得到硅胶片的应变分布。利用万能材料试验机对硅胶片进行实验和计算得到硅胶片的力学参数,根据力学参数建立有限元模型,并对硅胶片的张应力、张应变分布进行模拟计算。将实验结果和模拟结果进行比较。结果有限元结果和实验结果基本一致,张应力、张应变的最大值均出现在加载点处,中间区域应力、应变较为均匀。硅胶片中间60%面积区域可视为均匀应变场。结论研究结果可为后期血管壁内皮细胞的动态培养以及细胞力学研究提供实验技术。     

Study on Fluid Shear Flow on Osteoblast-like Cells in Their Tensile Loading Experiments

In vitro cell loading experiments are used to investigate stimulation of strain to cellular proliferation. As the flowing conditions of culture fluid in loading systems has been little known, strictly people can not detect the influence of strain to cellular proliferation exactly because shear flow can enhance cell proliferation either. Based on the working principle and cyclic loading parameters, we simplify Navier-Stokes equation to describe the flow of culture fluid on substrates of uniaxial and equi-biaxial fiat tensile loading systems and four point bending system. With approximate solutions, the distributions of velocity field and wall shear flow to cells are gained. Results show: shear flows are zero in the middle (or fixed point or line) of substrate for all systems, and they get larger proportionally to distance from middle and substrate elongate; the shear flow on the substrate of four point bending system is much greater than those of others. This shear flow in four point bending system, confirmed by Owan, I., et al with OPN mRNA increase in their experiment, could cause more influence to osteoblast-like cells than that caused by strain. We estimate the average magnitude of shear stress in Owan‘ s device, the results are consistent with other experimental data about shear flow. In conclusion our study makes it possible to differentiate the influence of strain on cellular proliferation to that of shear flow in loading experiments with the devices mentioned above quantitatively.     

Development of high-throughput perfusion-based microbioreactor platform capable of providing tunable dynamic tensile loading to cells and its application for the study of bovine articular chondrocytes

Mammalian cells are sensitive to extracellular microenvironments. In order to precisely explore the physiological responses of cells to tensile loading, a stable and well-defined culture condition is required. In this study, a high-throughput perfusion-based microbioreactor platform capable of providing dynamic equibiaxial tensile loading to the cultured cells under a steady culture condition was proposed. The mechanism of generating tensile stimulation to cells is based on the pneumatically-driven deformation of an elastic polydimethylsiloxan (PDMS) membrane which exerts tensile loading to the attached cells. By modulating the magnitude and frequency of the applied pneumatic pressure, various tensile loading can be generated in a controllable manner. In this study, the microbioreactor platform was designed with the aid of the experimentally-validated finite element (FE) analysis to ensure the loading of tensile strain to cells is uniform and definable. Based on this design, the quantitative relationship between the applied pneumatic pressure and the generated tensile strain on the PDMS membrane was established via FE analysis. Results demonstrated that the proposed device was able to generate the tensile strain range (0~0.12), which covers the physiological condition that articular chondrocytes experience tensile strain under human walking condition. In this study, moreover, the effect of tensile loading on the metabolic, biosynthetic and proliferation activities of articular chondrocytes was investigated. Results disclosed that the dynamic tensile loading of 0.12 strain at 1 Hz might significantly up-regulate the synthesis of glycosaminoglycans while such stimulation was found no significant influence on the metabolic activity, the synthesis of collagen, and the proliferation of chondrocytes. Overall, this study has presented a high throughput perfusion micro cell culture device that is suitable for precisely exploring the effect of tensile loading on cell physiology.     

基于超弹性模型的动脉血管数值计算

目的：实验研究表明。血管在周向与轴向两种单轴向拉伸作用下表现出不同的力学特性,本文通过对血管单轴拉伸的数值计算,给出分别适用于周向和轴向荷载的模拟方法。方法：基于超弹性本构模型对轴向和周向两种单轴拉伸作用下血管的应力一应变关系进行数值计算,并结合血管组织结构特点及模型适用范围对结果进行分析,同时通过数值计算对Holzapfel．Gasser-Ogden模型中的各向异性参数对结果的影响展开讨论。结果：计算结果显示单一使用各向同性超弹性应变势函数无法准确完整的模拟两种情况下的单轴拉伸实验,周向拉伸采用各向同性超弹性本构模型的数值结果较好的吻合实验,而轴向拉伸宜采用Holzapfel-Gasser-Ogden模型。Holzapfel。Gasser-Ogden模型中各向异性参数1描述血管中两组增强纤维主方向的分散程度,y值越大即纤维平均主方向与轴向加载方向夹角越小,在外荷载作用下越容易使得纤维旋转到荷载方向;参数K描述血管中每组增强纤维主方向上纤维的分散程度。K值越大,纤维在基体中分散越广泛,材料性子越接近纤维,宏观表现越硬。结论：本文基于超弹性本构模型对轴向和周向两种单轴拉伸作用下血管的应力应变关系进行数值计算,提出分别用多项式形式的各向同性超弹性本构模型数值计算周向荷载作用下应力应变关系、Holzapfel-Gasser-Ogden各向异性超弹性本构模型数值模拟轴向荷载下力学性质,数值结果与实验吻合较好,为心血管系统的数值模拟提供指导,对血管系统的力学机制和临床研究具有重要意义。     

Can human mesenchymal stem cells survive on a NiTi implant material subjected to cyclic loading?

Nickel-titanium shape memory alloys (NiTi-SMAs) exhibit mechanical and chemical properties which make them attractive candidate materials for various types of biomedical applications. However, the high nickel content of NiTi-SMAs may result in adverse tissue reactions, especially when they are considered for load-bearing implants. It is generally assumed that a protective titanium oxide layer separates the metallic alloy from its environment and that this explains the good biocompatibility of NiTi. Cyclic loading may result in failure of the protective oxide layer. The scientific objective of this work was to find out whether cyclic dynamic strain, in a range relevant for orthopedic implants, diminishes the biocompatibility of NiTi-SMAs. In order to analyze the biocompatibility of NiTi-SMA surfaces subjected to cyclic loading, NiTi-SMA tensile specimens were preloaded with mesenchymal stem cells, transferred to a sterile cell culture system and fixed to the pull rods of a tensile testing machine. Eighty-six thousand and four hundred strain cycles at 2% pseudoelastic strain were performed for a period of 24 h or 7 days. Cytokines (IL-6, IL-8 and VEGF) and nickel ion release were determined within the cell culture medium. Adherent cells on the tensile specimens were stained with calcein-AM and propidium iodide to determine cell viability. Dynamic loading of the tensile specimens did not influence the viability of adherent human mesenchymal stem cells (hMSCs) after 24 h or 7 days compared with the non-strained control. Dynamic cycles of loading and unloading did not affect nickel ion release from the tensile specimens. The release of IL-6 from hMSCs cultured under dynamic conditions was significantly higher after mechanical load (873 pg ml(-1)) compared with static conditions (323 pg ml(-1)). The present work demonstrates that a new type of mechanical in vitro cell culture experiment can provide information which previously could only be obtained in large animal experiments.     

Viscoelastic properties of the cervical spinal ligaments under fast strain-rate deformations

The mechanical response of ligaments under fast strain-rate deformations is a necessary input into computational models that are used for injury assessment. However, this information frequently is not available for the ligaments that are routinely injured in fast-rate loading scenarios. In the current study, experiments were conducted at fast strain rates for the cervical spinal ligaments: the anterior longitudinal ligament, the posterior longitudinal ligament and the ligamentum flavum. Bone-ligament-bone complexes at three spine levels were harvested for mechanical testing. Displacement-controlled sub-failure uniaxial tensile tests were performed in both load-relaxation and sinusoidal conditions. A nonlinear (separable) viscoelastic model was used to examine the experimental data. An unexpected result of the modeling was that the instantaneous elastic functions could be approximated as linear for these strain rates. A five-parameter model was sufficient to characterize the ligament viscoelastic responses and had good predictive capacity under different applied loading conditions.     

Is perception of upper body orientation based on the inertia tensor? Normogravity versus microgravity conditions

During lateral leg raising, a synergistic inclination of the supporting leg and trunk in the opposite direction to the leg movement is performed in order to preserve equilibrium. As first hypothesized by Pagano and Turvey (J Exp Psychol Hum Percept Perform, 1995, 21:1070–1087), the perception of limb orientation could be based on the orientation of the limbs inertia tensor. The purpose of this study was thus to explore whether the final upper body orientation (trunk inclination relative to vertical) depends on changes in the trunk inertia tensor. We imposed a loading condition, with total mass of 4 kg added to the subjects trunk in either a symmetrical or asymmetrical configuration. This changed the orientation of the trunk inertia tensor while keeping the total trunk mass constant. In order to separate any effects of the inertia tensor from the effects of gravitational torque, the experiment was carried out in normo- and microgravity. The results indicated that in normogravity the same final upper body orientation was maintained irrespective of the loading condition. In microgravity, regardless of loading conditions the same (but different from the normogravity) orientation of the upper body was achieved through different joint organizations: two joints (the hip and ankle joints of the supporting leg) in the asymmetrical loading condition, and one (hip) in the symmetrical loading condition. In order to determine whether the different orientations of the inertia tensor were perceived during the movement, the interjoint coordination was quantified by performing a principal components analysis (PCA) on the supporting and moving hips and on the supporting ankle joints. It was expected that different loading conditions would modify the principal component of the PCA. In normogravity, asymmetrical loading decreased the coupling between joints, while in microgravity a strong coupling was preserved whatever the loading condition. It was concluded that the trunk inertia tensor did not play a role during the lateral leg raising task because in spite of the absence of gravitational torque the final upper body orientation and the interjoint coupling were not influenced.     

Design and application of a test system for viscoelastic characterization of collagen gels

Characterization and control of the mechanical properties of the extracellular matrix are critical to the interpretation of results of in vitro studies of cultured tissues and cells and for the design of functional engineered constructs. In this work a viscoelastic tensile test system and custom culture chambers were developed and characterized. The system allowed quantification of strain as well as the stresses developed during cyclic viscoelastic material testing. Finite element analysis of the culture chambers indicated that the tensile strains near the actuated ends of the gel were greater than the strains experienced by material in the center of the culture chambers. However, the strain was uniformly distributed over the central substance of the gel, validating the assumption that a homogeneous strain state existed in the central region of the chamber. Viscoelastic testing was performed on collagen gels that were created with three different collagen concentrations. Results demonstrated that there was a significant increase in the dynamic stiffness of the gels with increasing equilibrium strain, collagen concentration, and frequency of applied strain. With increasing strain rate, the phase angle, representing the energy dissipated, dropped initially and then increased at higher rates. Mechanical testing of gels at different time intervals up to 7 days after polymerization demonstrated that the material properties remained stable when appropriate environmental conditions were maintained. The ability to characterize the viscoelastic properties of gels after different periods of culture will allow the quantification of alterations in gel material properties due to changes in cell cytoskeletal organization, cell-matrix interactions, and cellular activity on the matrix. Further, the test device provides a means to apply controlled mechanical loading to growing gel cultures. Finally, the results of this study will provide guidance to the design of further experiments on this substrate.     

成骨细胞的体外剪切应力加载实验的变形分析及信号转导区域探讨

目的 根据已有体外培养鼠成骨细胞的参数实验数据,估算剪切应力加载实验中细胞整体剪切形变,借以研究细胞的主要转导区域.方法 计算过程采用黏弹性力学理论,对细胞运用了标准黏弹性模型,并简化其膜所受剪切力为均匀.结果 细胞剪切力产生的细胞变形大约是引起成骨细胞相同生物学响应的拉伸加载变形的十分之一.结论 从细胞总的力学刺激生物学响应来看,剪切应力加载实验中细胞的整体变形所产生的力学转导是可以忽略的,主要转导区域在承受剪切应力的细胞膜.     

Longitudinal diffusion tensor imaging in a rat brain glioma model

In order to investigate the properties of water motion within and around brain tumors as a function of tumor growth, longitudinal diffusion tensor imaging (DTI) was carried out in a rat brain glioma (C6) model. As tumors grew in size, significant anisotropy of water diffusion was seen both within and around the tumor. The tissue water surrounding the tumor exhibited high planar anisotropy, as opposed to the linear anisotropy normally seen in white matter, indicating that cells were experiencing stress in a direction normal to the tumor border. When tumors were sufficiently large, significant anisotropy was also seen within the tumor because of longer-range organization of cancer cells within the tumor borders. These findings have important implications for diffusion-weighted MRI experiments examining tumor growth and response to therapy. Copyright (c) 2008 John Wiley & Sons, Ltd.