切应力对与血管平滑肌细胞联合培养的内皮细胞微管骨架重构的影响

Flow-mediated regulation of endothelial cell microtubule cytoskeleton in a coculture system

目的探讨切应力对与血管平滑肌细胞(VSMCs)联合培养的内皮细胞(ECs)中微管的聚集重构的影响,为阐明应力诱导血管重建的分子机制提供一些实验证据。方法应用ECs与VSMCs联合培养的平行平板流动腔系统,给ECs面施加15dyne/cm2的层流切应力,以静态条件下联合培养的ECs为对照组,用WesternBlot、免疫荧光细胞化学和图像分析等技术,研究切应力作用下与VSMCs联合培养的ECs的微管聚集的变化。结果静态联合培养组,ECs微管骨架的排列是稀疏、发散和无规律的。切应力诱导了ECs的微管的重构,,微管骨架变得有序,朝切应力的方向规律的排列。切应力能够促进ECs的微管聚集,与对照组相比,切应力作用下的ECs内多聚微管的数量增加,切应力作用3h,ECs内多聚微管的数量达到峰值,之后开始下降。结论切应力诱导和促进了EC的微管骨架发生重构(聚集)。结果提示:微管可能是机械应力刺激作用的靶标,应力可能通过它改变ECs的形态,影响细胞的黏附与迁移等功能。

Objective The influence of shear stress on the assembly of microtubules in endothelial cells （ECs） was elucidated in the co-culture system with vascular smooth muscle cells to provide some experimental evidences for molecular mechanisms of shear stress-induced vascular remodeling, Methods The assembly of microtubules in ECs co-cultured with vascular smooth muscle cells （VSMCs） under shear stressor 15dyne/cm^2- was examined by Western Blot, immunocytochemistry and image analysis. ECs were co-cultured with VSMCs under static conditions as a control. Results The microtubule arrangements in the control group showed sparsely, emanative and ruleless, After laminar shear stress （1.5Pa, 15dynes/cm^2） applied to ECs co-cultured with VSMCs for 12h, a distinct change in microtubule arrangement appeared to the regular state and oriented to the direction of shear stress, Application of shear stress to the co-cultured ECs also promoted the quantitative assembly of microtubules in ECs. The polymeric tubulin mass became decreased in ECs after exposed to shear stress for 3 hours. Conclusions Shear stress induces and promotes microtubule assembly in the co-cultured ECs. Furthermore, these data suggest that microtubules could be a potential target for translating changes in externally applied mechanical stimulation to alterations in cellular phenotype and functions such as adhesion and migration.