Study of roles of cytoskeletons in morphological responses of vascular endothelial cells to mechanical stimuli

1)应力纤维的力学性能评价采用本研究开发的微针拉伸试验系统对平滑肌细胞（SMCs）和内皮细胞（ECs）中提取的应力纤维的力学性能进行了测试。在力-应变曲线上观察到应变致硬化。我们还发现，提取的SFs在离开底物后缩短到原始长度的80%左右，这表明SFs在细胞质中已经存在菌株。将单个SFs从零应力长度拉伸回原始长度所需的力对于SMCs约为10 nN，对于ec约为4.1 nN。利用微加工技术，在硅衬底上制作了一个带有微针状柱阵列（直径3μm，高度10μm，间距8μm）的模具，以估计细胞的牵引力。SMCs在基质上的扩散产生了桩的偏转，并与肌动蛋白丝的应力纤维的组织有关。细胞间的牵引力差异很大，平均约为12毫安。3)微底物的循环拉伸试验我们使用带有微柱阵列的微结构底物对ECs进行循环拉伸，细胞在FAs之间选择性拉伸，但fa -底物接触面积几乎没有拉伸。在ECs循环拉伸3h后，平面和微柱基质上的细胞垂直于拉伸方向排列。拉伸6h后，微柱基板上的应力纤维向拉伸方向取向约90°，而平面基板上的应力纤维向拉伸方向取向约60°。这些结果表明，FA-substrate接触区域的应变可能会影响应力纤维在ec中响应循环拉伸的重定向速率。

1) Evaluation of mechanical property of stress fibersMechanical properties of stress fibers extracted from smooth muscle cells (SMCs) and endothelial cells (ECs) were measured with a microneedle-based tensile test system developed in this study. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN for SMCs and 4.1 nN for ECs.2) Measurement of traction forces of cells using microfabricated micropost arraysA mold of silicon substrates with arrays of micro-needle-like posts (3μm in diameter, 10μm in height, and 8μm intervals) was fabricated to estimate cellular traction forces using microfabrication techniques. SMCs spreading on the substrates produced deflection of the posts and was associated with organization of stress fibers of actin filaments. Traction forces varied considerably among cells, showing an average of approximately 12 nN.3) Cyclic stretch test with microsubstratesWe applied cyclic stretch to ECs using a microstructured substrate with arrays of micropost, on which cells were selectively stretched between FAs but FA-substrate contact area were hardly stretched. After exposure ECs to cyclic stretch for 3h, cells on both a flat and the micropost substrates aligned perpendicular to the direction of stretch. Stress fibers oriented about 60° to the stretch direction for the flat substrate at 3h, while stress fibers on the micropost substrate oriented approximately 90° to the stretch direction after 6h stretching. These results suggest that strain in FA-substrate contact area may have impact on rates of reorienation of stress fibers in ECs in response to cyclic stretch.