权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Bio-engineering study on the mechanism of endothelial cell responses to blood flow

内皮细胞对血流反应机制的生物工程研究

基本信息

批准号：
03404062
负责人：
KAMIYA Akira
金额：
$ 9.47万
依托单位：
Institute of Medical Electronics, Faculty of Medicine, Univ.of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1994
项目状态：
已结题

项目摘要

To elucidate the mechanism of vascular endothelial cell (EC) responses to blood flow, cultured ECs were exposed to controlled levels of shear stress in a flow-loading device and their responses were examined in vitro. Concretely, three different experiments were performed in this project ; 1) as regards the signal transduction of shear stress, changes in intracellular Ca^<++> concentration ([Ca^<++>]) were monitored by a calcium indicator, Fura-2, and fluorescence photomicroscopy when ECs were subjected to shear stress. 2) as to cell responses to shear stress, the production of nitric oxide, a potent vasodilator, by ECs was measured. 3) as to shear stress effect on gene expression, changes in the mRNA levels of adhesion molecules were analyzed with reverse transcriptase/polymerase chain reaction method. The results obtained are as follows.1) Application of medium flow increased [Ca^<++>] in ECs. The response needs the presence of extracellular ATP,and at 500 nM ATP,[Ca^<++>] increased in proportion to flow rate. Flow-loading experiments using two perfusates with different viscosities revealed that flow-induced [Ca^<++>] increase in shear stress-rather that shear rate-dependent.2) Shear stress stimulated nitric oxiide production by ECs.3) Shear stress decreased the mRNA level of vascular adhesion molecule-1 in mouse venule ECs, and at the same time down regulated the cell surface expression.These data suggest that vascular ECs can perceive shear stress generated by blood flow and alter their functions in both protein and gene levels, which is probably involved in flow-dependent vascular structuring and atherogenesis.

为了阐明血管内皮细胞（EC）对血流的反应机制，将培养的EC暴露于流量加载装置中的受控水平的剪切应力下，并在体外检测其反应。具体而言，本课题进行了三个不同的实验：1）关于切应力的信号转导，通过钙指示剂Fura-2和荧光显微镜监测内皮细胞在切应力作用下细胞内Ca^++浓度（[Ca^++]）的变化。2)至于细胞对剪切应力的反应，测定了内皮细胞产生一氧化氮（一种有效的血管扩张剂）的情况。3)采用逆转录聚合酶链反应（RT-PCR）方法分析剪切力对细胞粘附分子mRNA表达的影响。结果表明：（1）应用介质流可增加内皮细胞内[Ca^++]。该反应需要细胞外ATP的存在，并且在500 nM ATP下，[Ca^<++>]与流速成比例地增加。用两种不同粘度的灌流液进行的流量负荷实验表明，流量诱导的[Ca^++]增加与切应力有关，而不是与切应力有关。2）切应力刺激内皮细胞产生一氧化氮。3）切应力降低小鼠小静脉内皮细胞血管粘附分子-1的mRNA水平，这些结果表明，血管内皮细胞可以感知血流产生的切应力，并在蛋白和基因水平上改变其功能，其可能涉及流动依赖性血管结构化和动脉粥样硬化形成。