剪切力对脑微血管内皮细胞骨架蛋白的影响

FLUID SHEAR STRESS-INDUCED CYTOSKELETON CHANGES OF BRAIN MICROVASCULAR ENDOTHELIAL CELL MONOLAYERS IN RAT

利用自行研制的细胞流动小室对大鼠脑微血管内皮细胞在剪切力作用下细胞骨架蛋白的结构改变进行了初步研究，结果提示脑微血管内皮细胞在剪切力作用下，细胞形态学发生明显改变，细胞间隙增大、皱缩、脱落，细胞骨架蛋白的结构也有类似的变化，骨架蛋白沿流动方向重新排列，微丝中Ｆ－Ａｃｔｉｎ的数量增加、变粗。这些改变的直接后果是内皮细胞通透性的增加。该工作为进一步开展剪切力对微血管内皮细胞功能。

To determine the effects of fluid shear stress on microvascular endothelial cell monolayers, we designed a flow chamber to investigate the cytoskeleton changes after a period of shear stress effect. A coverslip carrying confluent rat brain microvascular endothelial cell monolayers was inserted into the device and the chamber gave parallel plate flow. The experiment proceeded with various perfusion rates. The perfusion medium was D-Hank's balanced salt solution with the viscosity of 0.75 mPa.s at 37℃. The shear stress effected on the monolayers were 0.17, 0.35, 0.70 and 1.41 dyn/cm2 by a roller pump for a perfusion period of 30min, 1h and 3h respectively. The whole device was brought to 37℃ with an air curtain incubator. After a period of perfusion, the coverslip was taken out and then stained with Coomassie BB and FITC-Phalloidin. The stained coverslip was placed on the stage of an inverted tissue culture microscope with camera. The monolayer photos were analyzed by image processing and analysis system. On all the shear stress level cytoskeleton showed the similar changes. Cytoskelleton were rearranged parallel to the direction of shear stress.Immunofluorescence showed that the number of F-Actin was significantly increased after a period of 30min. The intercellular space increased and monolayers shank. The results showed the significant rearrangement of cytoskeleton caused by shear stress and transfer from G-Action to F-Actin which indicated that the brain microvascular endothelial cell monolayers might be more sensitive to the fluid shear stress.