肌球蛋白轻链激酶在机械牵拉诱发人肺微血管内皮细胞通透性增加中的作用

目的 评价肌球蛋白轻链激酶在机械牵拉诱发人肺微血管内皮细胞(HPMVECs)通透性增加中的作用.方法 体外培养HPMVECs,随机分为3组(n=4),机械牵拉组(S组):单层细胞与RGD肽包被磁珠,在无血清MCDB131培养基中孵育2 h,经无血清培养基洗涤去除未结合磁珠后,给予磁扭力刺激(刺激频率3 Hz,刺激强度4.2 mT,刺激时间2 h);ML9处理组(M组):单层细胞与RGD肽包被磁珠,在无血清MCDB131培养基中孵育2 h,同时加入肌球蛋白轻链激酶抑制剂--ML9(50 μmol/L),其余处理同S组;对照组(C组):不给予磁扭力刺激,而将与S组相同的洗涤细胞放人细胞培养箱继续培养2 h.用transwell模型及荧光素标记的右旋糖苷检测HPMVECs通透性,免疫荧光法检测整合素αVβ3和肌动蛋白的分布.结果 与C组比较,S组HPMVECs通透性增加(P＜0.05),而M组差异无统计学意义(P ＞ 0.05);与S组比较,M组HPMVECs通透性降低(P＜0.05).S组HPM-VECs膜周边肌动蛋白逐渐消失,肌动蛋白聚集形成应力纤维,且整合素αVβ3明显簇积,C组和M组肌动蛋白多分布在HPMVECs膜周边,整合素αVβ3均匀分布在细胞表面.结论 机械牵拉诱发HPM-VECs通透性增加的机制与肌球蛋白轻链激酶活化介导的应力纤维形成及整合素αVβ3簇积有关.

Role of myosin light chain kinase in increase in permeability of human pulmonary microvascular endothelial cells induced by mechanical stretch

Objective To evaluate the role of myosin light chain kinase(MLCK)in the increase in the permeability of human pulmonary microvascular endothelial cells(HPMVECs)induced by mechanical stretch.Methods HPMVECs were cultured in vitro and then randomly divided into 3 groups(n = 4 each): mechanical stretch group(group S),ML9 treatment group(group M)and control group(group C).In group S,the monolayer cells and magnetic beads coated with an RGD peptide were incubated in serum-free MCDB131 medium for 2 h,the unbound beads were washed out,and then the cells were exposed to magnetic twisting stimulation(MTS)for 2 h(frequency 3 Hz,intensity 4.2 mT).In group M,the monolayer cells and magnetic beads coated with an RGD peptide were incubated for 2 h in serum-free MCDB131 medium in which MLCK inhibitor ML9(50μmol/L)was added,and the other procedures were the same as in group S.In group C,the cells after washing as in group S were incubated for 2 h without exposing to MTS.Monolayer permeability was detected with FITC-dextran flux in transwell model.The distribution of integrin αVβ3 and actin was detected using the immunofluorescence assay.Results Compared with group C,the permeability of HPMVECs was significantly increased in group S,while no significant change was found in group M.Compared with group S,the permeability of HPMVECs was significantly decreased in group M.In addition,actin polymerized to form stress fiber,and integrin αVβ3 clustered at the end of stress fiber in group S,while actin mainly distributed in the surrounding area of the cell membrane and integrin αVβ3 evenly distributed on the cell surface in group C and M.Conclusion The mechanism by which mechanical stretch induces the increase in the permeability of HPMVECs is related to MLCK activation-mediated stress fiber formation and integrin αVβ3 clustering.