流体剪切力和细胞骨架阻断对肌动蛋白丝的影响

目的：探讨流体剪切力对成骨细胞细胞骨架的影响.方法：实验分为3组,Ⅰ组（流体剪切力）,Ⅱ组（流体剪切力＋细胞松弛素D,Cytochalasin D）,Ⅲ组（流体剪切力＋噻氨酯哒唑,Nocodazole）.分别对Ⅰ,Ⅱ,Ⅲ组施加12dyne/cm2流体剪应力,应力作用时间分别为：0,10,30,60min.采用激光共聚焦显微镜、免疫荧光显微镜技术和免疫荧光染色方法对小鼠成骨细胞骨架进行形态学观察、F-肌动蛋白表达的荧光定量分析.结果：细胞在不同时间点受到同一剪切应力后,细胞的排列方向发生改变,细胞内的F-肌动蛋白排列同应力方向一致,随着应力时间延长到1h,F-肌动蛋白变得厚而丰富,F-肌动蛋白的荧光强度同0时段剪切力组相比明显增强（P〈0.01）.当加入细胞松驰素D后,细胞内F-肌动蛋白结构发生改变,在力的作用下,细胞内微丝断裂明显,随着拉伸时间的延长,微丝断裂更为明显,F-肌动蛋白的荧光强度同0时段剪切力组相比显著减弱（P〈0.01）.加入噻氨酯哒唑后,在剪切力作用下细胞内微管断裂,F-肌动蛋白丝完整且边缘呈锯齿状改变,它的荧光强度同0时段剪切力组相比也有明显减弱（P〈0.05）.结论：不同时间点受到同一剪切应力对细胞骨架微丝、微管结构产生一定的影响,微丝解聚和重排以及微管断裂与否在细胞力传导中起重要作用.

Effects of fluid shear stress and cytoskeletal disruption on the actin cytoskeleton

AIM：To investigate the effects of fluid shear stress on cytoskeleton of osteoblasts,we examined the effects applying confocal laser scanning microscope and immunofluorescence microscopy.METHODS：The experiments were divided into 3 groups,Ⅰgroup,fluid shear stress;Ⅱgroup,fluid shear stress ＋cytochalasinD（CD）;Ⅲ group,fluid shear stress＋nocodazole（NO）.12 dyne/cm2 fluid shear stress was applied on the osteoblasts at four time points,and cells was marked by FITC-phalloidin and PI double Iabel technique.Images were observed by confocal laser scanning microscope and immunofluorescence microscopy.RESULTS：After the application of fluid shear stress for 1 h at 12 dnye/cm2,F-actin filaments were organized into stress fibers that became thicker and more abundant than 0 min fluid shear stress cells.Shear stress was associated with significant increases of fluorescence intensity of F-actin.The minimum doses of nocodazole（microtubules）and cytochalasin D（actin filaments）partially disrupted the cytoskeleton.In cytochalasin D-treated cultures,F-actin filaments and the fluorescence intensity of F-actin significant decreased.While,F-actin slightly decreased in nocodazole treatment.CONCLUSION：These results indicate that the cytoskeleton,especially F-actin filaments,play a crucial role in cellular mechanotransduction.