Effects of derection and pulsatility of flow on morphology and functions of cultured endothelial cells

研究了不同流动方向对培养的牛主动脉内皮细胞形态和细胞骨架结构的影响。将培养于玻璃上的完全融合的内皮细胞置于20dyne/cm2的流体剪切应力作用下24小时，采用平行平板流动室，低截面高0.5 mm，宽60 mm。对(I)单向流动和(Ii)间隔30分钟的往复流动进行了剪切流暴露实验。在往复流动实验中，通过滚柱泵和定时器控制夹管阀交替改变介质流量。流暴露后固定内皮细胞，用罗丹明鬼臼乙素对FF-肌动蛋白细丝进行染色。在装有荧光光学装置的显微镜下观察和拍摄内皮细胞，并在打印的光学显微镜上测量每个细胞的形状指数。两种流动条件下均可观察到沿流动方向排列的细胞和应力纤维的形成。单向流动条件下的细胞排列程度高于往复流动条件下的细胞排列程度。内皮细胞在往复流动条件下的形态指数(0.68±0.12)介于无流动(0.87±0.06)和单向流动(0.48±0.15)之间。在静态培养的细胞中，F-肌动蛋白细丝形成致密的周边带，呈蜂窝状。在单向流动条件下，应力纤维主要形成在细胞的中心区域，并与流动方向一致。每个细胞上游周围的F-肌动蛋白细丝染色较深。相反，在往复流动中暴露的细胞中的F-肌动蛋白细丝几乎均匀染色。众所周知，内皮细胞对剪切力有反应。此外，总而言之，我们的实验结果表明，内皮细胞

Effects of flow direction on shape and cytoskeletal structure of cultured bovine aortic endothelial cells were studied. Fully confluent endothelial cells cultured on glass were subjected to fluid-imposed shear stress of 20 dyne/cm^2 for 24 hours using a parallel plate flow chamber which has a low section 0.5 mm in height and 60 mm in width. Experiments on shear flow exposure were performed for (i) one-way flow, and (ii) reciprocating flow with a 30 minute interval. In the reciprocating flow experiment, medium flow was alternatively changed in the oposite direction by a roller pump and pinch valves controlled with a timer. After the flow exposure endothelial cells were fixed and Ff-actin filaments were stained with rhodamine phalloidin. Endothelial cells were observed and photographed by means of a microscope equipped with epiflluorescence optics, and the shape index of each individual cell was measured on the printed photomicrographs. Cells aligned with the flow direction and stress fibers were observed to be developed under the both flow conditions. The degrees of cell alignment under the one-way flow conditon became higher than those under reciprocating flow. Shape index value of endothelial cells exposed to the reciprocating flow (0.68(〕SY.+-.〔)0.12) was between that of the no-flow (0.87(〕SY.+-.〔)0.06)and the one-way flow conditions (0.48(〕SY.+-.〔)0.15). Dense peripheral bands of F-actin filaments were characteristically formed in honeycomb shape in the statically cultured cells. Under the one-way flow conditions, stress fibers were mainly formed in the central area of the cells and aligned with the flow direction. And the F-actin filaments around the upstream side of each cell were observed to be strongly stained. In contrast, F-actin filaments in the cells exposed to reciprocating flow almost stained homogeneously. Endothelial cells are well known to respond to shear stress. Furthermore, in conclusion, our experimental results show that endothelial cell