梯度切应力对血管内皮细胞增殖、迁移及Connexin43表达的影响

目的:研究流体切应力空间梯度和均匀切应力对血管内皮细胞(Ecs)增殖、迁移和对Connexin43表达的影响,为阐明切应力诱导支架内再狭窄分子机制提供一些实验依据。方法:将脐静脉内皮细胞置于平行平板流动腔系统,分别施加11dyn/cm2的均匀切应力(矩形组)和15～6.6dyn/cm2范围、梯度分别为1.5 dyn/cm3和3 dyn/cm3的切应力,加载时间为6 h、12 h、24 h,以静态条件下培养的Ecs为对照组(静止组)。检测梯度切应力对Ecs增殖、迁移能力的影响及免疫印迹法(Western Blotting)检测6 h、12 h和24 h的定量表达情况。结果:①梯度切应力受力组的EC在受力6 h后进入S期与G2+M期的细胞明显多于静止组与矩形受力组(P<0.05)。②同静止组和矩形受力组相比,切应力梯度组ECs 24 h的迁移(68±15vs35±4vs33±7,P<0.01,n=9)能力显著提高。③免疫印迹结果显示:同矩形受力和静止组相比,Ecs受切应力梯度作用受力6 h开始Connexin43表达水平就显著增高(P<0.01,P<0.05),且这种较高的表达水平延续至24 h。结论:切应力通过上调Con-nexin43表达促进了Ecs增殖、迁移,提示connexin信号通路参与了切应力条件下Ecs增殖、迁移过程的信号转导。

Effects of gradient shear stress on vascular endothelial cell proliferation and migration and connexin43 expression

Objective：To observe the cell proliferation,migration and connexin43 expression in human umbilical vein endothelial cells（HUVEC） after gradient shear stress and steady flow shear stress stimulation for the experimental foundation with the molecular mechanisms of shear stress-induced in-stent restenosis.Methods：The endothelial cells were subjected to different fluid shear stress in the flow chamber and divided into three different experimental groups： static group,gradient shear stress group（15～6.6 dyn/cm2） and steady flow shear stress group（1ldyn/cm2）.The effects of shear stress on the migration,proliferation of ECs,expression of connexin43 at 6 h,12 h and 24 h,respectively,was examined by Western Blotting.Results：①The amount of HUVEC entering cell cycle phase of S and G2＋M in gradient shear stress group was much greater than that in two other groups（P0.05）.②Compared with the two other groups at 24 h,Ecs migration was significantly promoted under gradient shear stress（68±15 vs.35±4 vs.33±7,P0.01,n=9）;③ Western blotting demonstrated that connexin43 expression was significantly increased in gradient shear stress group at 6 h as compared with the two other groups（P0.01,P0.05）and the higher expression level proceeded to 24 h.Conclusion：Connexin43 activation could be induced by shear stress.The findings suggest that connexin may be involved in the signaling pathway to promote Ecs migration and proliferation.pathway.