EFFECT OF MECHANICAL FORCES ON VASCULAR CELLS

这项资助申请的长期目标是了解 细胞对机械信号的反应机制， 一部分直接的体内环境。 我们已经开发了一个 使我们能够精确地进行双轴变形的装置 并评估这种机械应变的影响 形态学、生化学和生理学上。 我们建议 机械应变改变细胞功能， 适应其机械环境可能损害细胞功能， 最终细胞存活。 使用该装置，我们建议：1） 确定基质蛋白合成和基质基因表达是否 由于施加双轴应变而改变，以及2），以确定 内皮细胞机械化学转导机制 对双轴伸长的反应。 为了实现目标1，我们将分析 蛋白质和RNA水平的基质合成，特别强调 对纤维连接蛋白和多种形式的剪接产生的 主要的成绩单。 为了实现第二个目标，我们将测量 与机械刺激相关的细胞内瞬变， 开发钙动力学分析模型，重点是 由于受伤而导致的钙积累。 我们还将确定， 磷酸化事件与机械转导过程有关 在内皮细胞中。 基质蛋白合成的变化将是 通过放射性同位素掺入研究评估，聚丙烯酰胺凝胶 同位素标记的蛋白质的电泳， 标记的蛋白质，Western印迹和ELISA。 对基质基因的影响 将使用特异性基质蛋白cDNA探针评估表达 胶原蛋白和纤连蛋白同种型。 这些探针将用于 细胞RNA的北方杂交和狭缝印迹分析 已经双轴应变不同时期的培养物， 时间 此外，来自双轴应变培养物的RNA将被扩增， 并使用聚合酶链反应技术定量。 磷酸化事件是由细胞的双轴变形引起的， 文化的特点将是为了了解 细胞对施加的机械力的反应机制。 我们将 我也试图测量细胞内离子瞬变与 使用特异性荧光染料的机械刺激， 细胞内自由调用化学计量。 这些研究的目的是 以确定存在于血管细胞中的机械传导系统， 定义细胞对机械力反应的分子机制 力.

The long term goal of this grant application Is to understand the mechanism by which cells respond to mechanical signals which are a part of their immediate in vivo environment. We have developed an apparatus which permits us to apply precise levels of biaxial deformation to cells in culture and to assess the effects of this mechanical strain morphologically, biochemically and physiologically. We propose that mechanical strain modifies cell function and that the cell's ability to acclimate to its mechanical environment may compromise cell function and ultimately cell survival. Using this apparatus, we propose to: 1) determine If matrix protein synthesis and matrix gene expression are altered as a result of applied biaxial strain and 2), to determine a mechanism of mechanochemical transduction in endothelial cells In response to biaxial elongation. To accomplish aim 1, we will analyze matrix synthesis at the protein and RNA levels with particular emphasis on the fibronectin and the multiple forms which arise from splicing of the primary transcript. To accomplish the second aim, we will measure the Intracellular transients associated with mechanical stimulation and develop an analytical model for the calcium kinetics with emphasis on calcium accumulation as a result of injury. We will also determine if phosphorylation events are involved with the mechano-transduction process in endothelial cells. Changes in matrix protein synthesis will be assessed by radioisotope incorporation studies, polyacrylamide gel electrophoresis of isotopically labeled proteins, protease digestion of labeled proteins, Western blotting and ELISAs. Effects on matrix gene expression will be evaluated using specific matrix protein cDNA probes for collagens and fibronectin isoforms. These probes will be used in Northern hybridization and slot blot analysis of RNAs extracted from cell cultures which have been biaxially strained for different periods of time. In addition, RNAs from biaxially strained cultures will amplified and quantitated using polymerase chain reaction technology. Phosphorylation events which result from biaxial deformation of cells in culture will be characterized with a view toward understanding the mechanism of cellular response to applied mechanical forces. We will also attempt to measure the Intracellular ion transients associated with mechanical stimulation using specific fluorescent dyes which bind intracellular free Call stoichiometrically. The goal of these studies is to define the mechanotransduction system present in vascular cells and to define the molecular mechanism of cellular response to applied mechanical forces.