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

1)应力纤维力学性能的评价本研究利用自行研制的微针拉伸测试系统，测定了从血管内皮细胞(ECs)和平滑肌细胞(SMC)中提取的应力纤维的力学性能。在力-应变曲线上观察到应变诱导的硬化现象。我们还发现，提取的SFS从底物上移出后，长度缩短到原来的80%左右，这表明SFS在细胞质中有预先存在的菌株。将单个SFs从零应力长度拉伸到原始长度所需的力，SMCS约为10nN，ECS约为4.1nN。2)使用微制微柱阵列测量细胞牵引力利用微制造技术制作了具有微针状柱阵列的硅衬底模型(直径3μm，高度10μm，间隔8μm)，以估计细胞牵引力。铺展在基质上的SMC导致柱子的偏转，并与肌动蛋白细丝的应力纤维的组织有关。细胞间牵引力差异很大，平均约12 nN。3)微基质循环拉伸试验我们使用微结构基质和微柱阵列对内皮细胞进行循环拉伸，细胞选择性地在FA之间拉伸，但FA-基质接触区域几乎不拉伸。将内皮细胞置于循环拉伸3h后，平板和微柱上的细胞垂直于拉伸方向排列。拉伸3h时，扁平基托的应力纤维与拉伸方向成60°左右，而微桩基托在拉伸6h时，应力纤维与拉伸方向成90°左右。这些结果表明，FA-衬底接触区的应变可能会影响循环拉伸时内皮细胞中应力纤维的重定向速率。

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.