Vascular Endothelial Cell Response to Mechanical Stimuli

由于今年是该项目的最后一期，我们从全球的角度审查了过去两年取得的成果，并决定开发一些新的实验方法来克服以前研究中的方法学问题1。一种观察活细胞内肌动蛋白细丝的新技术的发展被广泛用于观察细胞内肌动蛋白细丝的免疫荧光技术需要固定细胞，而且很难观察完整细胞中肌动蛋白细丝的动态变化。为了克服这个问题，我们将肌动蛋白细丝和绿色荧光蛋白(GFP)的桥梁元件α-肌动蛋白的组合基因引入血管内皮细胞。引入这种基因的细胞在它们的α-肌动蛋白中有自发荧光，因此它们的肌动蛋白细丝也有自发荧光。我们成功地观察到细胞在静态和剪切暴露条件下肌动蛋白细丝的动态变化。血管内皮细胞载流系统的研制动物实验结果与培养内皮细胞的结果有许多矛盾之处。为了建立一种弥补这两种实验缺点的新方法，我们开发了一种新的流量加载系统，在动脉段的内皮表面施加剪应力，在该系统中，切开的动脉段的一段平放，使其管腔内表面暴露在平行平板腔中流动。我们对兔胸主动脉施加垂直于血管轴的1pA剪应力，发现大多数内皮细胞在72小时内改变了其排列方向，平行于血流方向。该系统可广泛应用于未来血管内皮细胞对剪应力的力学响应研究。

As this year was the last term of this project, we examined our results obtained for the past two years form global point of view, and decided to develop some new experimental methods to overcome methodological problems in the previous studies.1. DEVELOPMENT OF A NEW TECHNIQUE TO OBSERVE ACTIN FILAMENTS IN ALIVE CELLSImmunofluorescence technique which has been utilized widely to observe intracellular actin filaments requires fixation of the cells, and it was vey difficult to observe actin filament dynamics in intact cells. To overcome this problem, we have introduced a combined gene of alpha-actinin, a bridging element of actin filaments and GFP (Green Fluorescent Protein) into vascular endothelial cells. Cells introduced such genes have autofluorescence in their alpha-actinin, and thus their actin filaments. We successfully observed actin filament dynamics in cells under static and shear-exposed conditions.2. DEVELOPMENT OF A FLOW-LOADING SYSTEM FOR ENDOTHELIAL CELLS ON ARTERIAL INTRALUMINAL SURFACEThere have been a lot of contradictions between results obtained from animal experiments and that based on cultured endothelial cells. To establish a new method compensating shortcomings of both experiments, we have developed a new flow-loading system to impose shear stress on the endothelial surface of an arterial segment, in which a piece of a cut-opened arterial segment is laid flat to expose its intraluminal surface to flow in a parallel plate chamber. We applied 1 Pa of shear stress to rabbit thoracic aortas perpendicularly to their vessel axis, and found that most of the endothelial cells changed their alignment parallel to the direction of flow in 72 hours. We concluded that this system can be widely used for the future studies on the mechanical response of vascular endothelial cells to shear stress.