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自行研制的微针拉伸试验系统，对平滑肌细胞（SMC）和内皮细胞（ECs）应力纤维的力学性能进行了测试。在力-应变曲线中观察到应变诱导硬化。我们还发现，提取的SFs缩短到约80%的原始长度后，他们从基板上脱落，这表明SFs有预先存在的应变在细胞质中。将单个SF从零应力长度拉伸回原始长度所需的力对于SMC为约10 nN，对于EC为约4.1 nN。2）使用微制造微柱阵列测量细胞的牵引力具有微针状柱阵列的硅衬底的模具（直径3μm，高度10μm，间隔8μm），以使用微制造技术估计细胞牵引力。SMC在基板上传播产生偏转的职位，并与肌动蛋白丝的应力纤维的组织。牵引力在细胞之间变化很大，显示平均值约为12 nN。3）微基底的循环拉伸试验我们使用具有微柱阵列的微结构基底对EC施加循环拉伸，在微柱阵列上，细胞在FA之间选择性地拉伸，但FA-基底接触区域几乎不拉伸。在将EC暴露于周期性拉伸3 h后，平板和微柱基底上的细胞均垂直于拉伸方向排列。对于平坦基底，在3 h时应力纤维取向为与拉伸方向成约60°，而微柱基底上的应力纤维在6 h拉伸后取向为与拉伸方向成约90°。这些结果表明，在FA-基板接触面积的应变可能有影响的应力纤维在ECs响应于循环拉伸的再取向率。

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.