周期性机械牵张对肺泡上皮细胞埃兹蛋白磷酸化的影响

Effects of cyclical mechanical stretch on the phosphorylation of ezrin in alveolar epithelium

目的探讨周期性机械牵张对人肺泡上皮细胞(A549)埃兹蛋白(ezrin)磷酸化的影响。方法经鉴定后将A549细胞培养在Ⅰ型胶原包被的Bioflex六孔板上,随机分组后进行机械牵张强度试验(给A549细胞施加5%、10%和20%3个不同强度的周期性机械牵张应力,牵拉4 h)和时相试验(给A549细胞施加强度为20%的周期性机械牵张应力,分别牵拉1、2、4、8和12 h),每个试验均设对照组,对照组不行牵拉。应用Western blot技术检测细胞内ezrin及p-ezrin蛋白的表达情况,以β-actin作为内参。结果(1)对照组A549细胞中ezrin和p-ezrin蛋白均有少量表达,与对照组相比,周期性机械牵张对ezrin蛋白表达差异无统计学意义(P>0.05)。(2)应力大小试验中,与对照组相比,5%牵张强度对p-ezrin蛋白表达差异无统计学意义;牵张强度从10%开始,p-ezrin蛋白表达随牵张应力呈强度依赖性增加,差异有统计学意义(P<0.05)。(3)在施加20%机械牵张应力下,p-ezrin表达量表现为牵拉时间依赖性且差异有统计学意义(P<0.05)。结论周期性机械牵张诱导A549细胞ezrin蛋白磷酸化,其可能参与了周期性机械牵张诱导肺泡上皮细胞损伤的调控过程。

Objective To investigate the effect of cyclical mechanical stretch on phosphorylaton of ezrin in human alveolar epithelium （A549）. Methods A549 cells were cultured in type I collagen - coated six - well Bioflex elastic silicone membranes and randomly grouped after being identified for follow study. For strain strength experiment （A549 cells were exposed to cyclic mechanical strain of 5%, 10% and 20% for 4 h） and time course experiment （A549 cells were subjected to cyclic mechanical strain of 20% for 1 h, 2 h, 4 h, 8 h and 12 h）. Western blot was used to detect the expression of ezrin protein and p - ezrin protein. Results Ezrin and p - ezrin were expressed in A549 cells under normal condition. Compared with control group, cyclic mechanical strain has no significant effects on the expression of ezrin pro- tein. The expression of p - ezrin induced by cyclic mechanical stretch was increased as strain intensified when cells were exposed to 10% or 20% strain, but 5% strain was not enough to induce ezrin phosphorylation. The p- ezrin induced by cyclic mechanical strain was increased in a time - dependant manner. Conclusion cyclical mechanical stretch induces ezrin protein phosphorylafion, which may be participated in the alveolus epithelium injury induced by cyclical mechanical stretch.