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向于流动方向，这取决于剪切应力的水平和暴露的持续时间。众所周知，细胞骨架成分，特别是f -肌动蛋白丝，在细胞形状对机械环境的适应过程中起着重要作用。在这个项目中，我们主要关注了两个主题，得到了如下结果：1.活细胞中肌动蛋白丝的动态行为我们观察了活内皮细胞在剪切应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)/肌动蛋白融合载体导入细胞。倒置荧光显微镜下通过CCD相机观察了引入细胞松弛素D后内皮细胞内肌动蛋白丝的动态行为。在不同的实验中，内皮细胞在平行板流室中受到2 Pa的流体剪切应力，并观察到细胞骨架结构。首先位于外周区域的肌动蛋白丝在流动暴露后似乎改变了排列，并与细胞的长轴对齐，虽然还没有朝向流动方向，但已经拉长了。采用流体流动-结构分析（即耦合场分析）模拟内皮细胞在剪切应力作用下的三维应力分布。在原子力显微镜测量细胞表面几何形状的基础上，建立了三维有限元模型。模型由一个流体单元和一个实体单元组成，分别代表流场和内皮细胞。对池内应力分布的分析结果表明，池内的上游和下游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 -肌动蛋白的分布密切相关。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