机械牵拉对人肺微血管内皮细胞单层通透性、F-肌动蛋白变化的影响

Effect of mechanical stretch on changes of monolayer permeability and F-actin in human pulmonary endothelial cells

目的:探讨机械牵拉后人肺微血管内皮细胞(human pul monary microvascular endothelial cells,HPMVECs)单层通透性、F-肌动蛋白(F-actin)的变化及它们之间的关系,并探讨Rho激酶抑制剂Y27632对上述变化的影响。方法:体外培养HPMVECs,磁扭力刺激(magnetic twisting sti mulation,MTS)实施机械牵拉,随机分为3组,对照组(C组):细胞不经MTS,在细胞培养箱中培养1h、2h;机械牵拉组(M组):MTS1h、2h;抑制剂组(Y组):细胞与Rho激酶抑制剂Y27632(10μmol/L)孵育2h后给予MTS1h、2h;用FITC标记的右旋糖苷在transwell模型中的通透率检测HPMVECs单层通透性,用免疫荧光染色检测HPMVECs的F-actin分布。结果:M组的HPMVECs单层通透性较C组显著增加(P<0.05),且MTS2h后HPMVECs单层通透性较MTS1h高(P<0.05);Y组则较M组显著降低(P<0.05)。C组细胞F-actin主要分布在细胞边缘;而MTS引起F-actin聚合形成应力纤维,而以刺激2h明显;而Y组应力纤维形成较M组明显减少。结论:机械牵拉可引起HPMVECs的单层通透性增高,F-actin聚集形成应力纤维,且HPMVECs单层通透性增高的机制可能与F-actin聚集成应力纤维密切相关。Y27632可通过抑制应力纤维形成而减轻机械牵拉对HPMVECs单层通透性的影响。

Objective：To investigate the changes of monolayer permeability and F-actin of human pulmonary microvascular endothelial cells（HPMVECs） induced by mechanical stretch with magnetic twisting stimulation（MTS）,and to study the influence of Rho kinase inhibitor Y27632.Method：HPMVECs were cultured in vitro and divided randomly into 3 groups：Control group（group C）,mechanical strething group（group M） and Y27632 group（group Y）.In group C,HPMVECs were kept in incubator for 1 h or 2 h without mechanical stretch with MTS;in group M,HPMVECs were mechanical stretched with MTS for 1 h or 2 h;in group Y,HPMVECs were incubated with Rho kinase inhibitor Y27632（10 μmol/L） for 2 h and then mechanical stretched with MTS for 1 h or 2 h.The monolayer permeability were observed using diffusion of FITC-dextran across the monolayer with transwell model.The F-actin distribution was evaluated by immunofluorescent staining.Result：Exposure to MTS for 1 h and 2 h significantly increased the permeability of HPMVECs monolayer compared with that of group C（P0.05）;permeability of HPMVECs monolayer with 2 h-MTS was higher than that of 1 h-MTS（P0.05）;the permeability of group Y was significantly lower than that of group M（P0.05）.Exposure to MTS for 1 and 2 h significantly induced polymerization of F-actin to form stress fiber compared with that in group C just with F-actin on the cell border,especially after 2 h-MTS;and the stress fiber of group Y was significantly less than that of the group M.Conclusion：Mechanical stretch with MTS can increase the permeability of HPMVECs monolayer and polymerize F-actin to form stress fiber;the increase of permeability might be related to the polymerization of F-actin.Y27632 can relieve permeability changes induced by mechanical stretch in HPMVECs by inhibiting stress fiber formation.