Development of Atomic Force Microscope System Combined with Confocal Laser Scanning Microscope to Simultaneously Measure Mechanical Stiffness and Observe Microstructure of Cultured Cells

在流动条件下，内皮细胞被拉长，并取决于剪切应力水平和暴露持续时间而定向于流动方向。细胞骨架成分，特别是F-肌动蛋白丝，在细胞形状适应力学环境的过程中起着重要的作用。在本项目中，我们主要围绕两个主题进行研究，并取得了如下成果.活细胞中肌动蛋白丝的动态行为我们观察了培养的活内皮细胞在剪切力作用下肌动蛋白丝的动态行为。为此，使用脂质体将绿色荧光蛋白（GFP）/肌动蛋白融合体的载体引入细胞中。在倒置荧光显微镜下，用CCD摄像机观察了细胞松弛素D（cytochalasinD）诱导内皮细胞内肌动蛋白丝的动态变化。在不同的实验中，内皮细胞在平行板流动室中暴露于2 Pa的流体剪切应力， ...更多信息 观察细胞骨架结构。首先位于周边区域的肌动蛋白丝似乎改变了流动暴露后的排列，并与细胞的长轴对齐，该长轴尚未定向于流动方向，但已经伸长.进行流体流动-结构分析，即耦合场分析，以模拟暴露于剪切应力的内皮细胞中的三维应力分布。三维有限元模型的基础上产生的细胞表面的几何形状通过原子力显微镜测量。该模型包括一个流体单元和一个固体单元，分别代表流场和内皮细胞。分析结果表明，在上游侧和下游侧都出现了较高的压应力。这些结果可能表明，细胞内的应力分布与F-actin的分布有密切的相关性。少

Under flow condition endothelial cells are elongated and oriented to flow direction depending upon the level of shear stress and the duration of exposure. It is well known that cytoskeletal components, especially F-actin filaments, are playing important roles in the process of adaptation of cell shape to mechanical environment. In this project We have mainly focued two topics and obtained the results shown below.1. Dynamic behavior of actin filament in living cellsWe observed dynamic behavior of actin filaments in living cultured endothelial cells during exposure to shear stress. To do this, a vector of a green fluorescent protein (GFP)/actin fusion was introduced into cells using a lipofectoamine. Dynamic behavior of actin filament in an endothelial cell after introducing cytochalasin D was observed through CCD camera under an inverted fluorescent microscope. In different experiments, the endothelial cells were exposed to fluid shear stress of 2 Pa in a parallel plate flow chamber and … More the cytoskeletal structure was observed. Actin filaments located first at peripheral regions seemed to change the alignment after flow exposure and aligned to a long axis of the cell, which was not oriented to the flow direction yet, but already elongated.2. Stress analysis in a cell exposed to shear stressA fluid flow-structural analysis, i.e. a coupled field analysis, was performed to simulate three dimensional stress distribution in endothelial cells exposed to shear stress. The three-dimensional finite element model was generated on the basis of the cell surface geometry measured by an atomic force microscopy. The model consisted of a fluid element and a solid element representing the flow field and the endothelial cells, respectively. Analytical results on stress distribution in the cell showed that high compressive stress appeared both in the upstream side and the downstream side. These results may indicate that the stress distributions in the cells have close correlation with the F-actin distributions. Less