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

为了阐明血管内皮细胞 (EC) 对血流反应的机制，将培养的 EC 暴露在流量加载装置中受控水平的剪切应力下，并在体外检查它们的反应。具体来说，该项目进行了三个不同的实验； 1)关于剪切应力的信号转导，当EC受到剪切应力时，通过钙指示剂Fura-2和荧光显微术监测细胞内Ca^<>浓度([Ca^<>])的变化。 2) 关于细胞对剪切应力的反应，测量了 EC 产生的一氧化氮（一种有效的血管舒张剂）。 3)对于剪切应力对基因表达的影响，采用逆转录酶/聚合酶链式反应方法分析粘附分子mRNA水平的变化。所得结果如下： 1)应用培养基流量增加了ECs中的[Ca^< >]。该反应需要细胞外 ATP 的存在，并且在 500 nM ATP 下，[Ca^< >] 与流速成比例增加。使用两种不同粘度的灌流液进行的流动加载实验表明，流动诱导的[Ca^<>]剪切应力增加，而不是剪切速率依赖性。2）剪切应力刺激ECs产生一氧化氮。3）剪切应力降低了小鼠小静脉ECs中血管粘附分子-1的mRNA水平，同时下调了细胞表面的表达。这些数据表明血管ECs可以感知血流产生的剪切应力并改变其在血管内皮细胞中的功能。蛋白质和基因水平，这可能与血流依赖性血管结构和动脉粥样硬化有关。

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.