细胞外信号调节激酶通路对人动脉硬化平滑肌细胞增殖、表型标志蛋白及微小RNA-31表达水平的影响

Effect of extracellular signal-regulated kinase pathway on proliferation, phenotype and microRNA-31 expression on human arteriosclerotic smooth muscle cells

目的 探讨阻断人动脉硬化平滑肌细胞中细胞外信号调节激酶（ERK）通路,进一步明确ERK、微小RNA（miR）-31与血小板源性生长因子（PDGF）-BB介导的平滑肌增殖之间的调控关系.方法 收集下肢动脉硬化闭塞患者下肢截肢标本,用贴壁法原代培养平滑肌细胞.相同代数的第3～6代血管平滑肌细胞（VSMCs）,加入PDGF-BB或PDGF-BB＋ PD98059分别用细胞计数试剂盒（CCK-8）法、Transwell法、Western blot、实时定量聚合酶链反应（Real-time PCR）检测VSMCs增殖、迁移能力、表型标志性蛋白表达以及miR-31表达水平.结果 经组织贴壁法成功培养出原代平滑肌细胞.PDGF-BB诱导动脉硬化细胞的增殖率上调102％ （P＜0.05）和迁移率上调246％ （P＜0.05）,ERK阻断剂PD98059能阻滞PDGF-BB诱导的诱导作用,平滑肌细胞增殖抑制53％（P＜0.05）和迁移抑制19％ （P ＜0.05）.与空白组比较PDGF-BB处理后,收缩表型标志蛋白α-平滑肌肌动蛋白（α-SMA）含量下调38％ （P ＜0.05）,增殖型蛋白骨桥蛋白（OPN）和miR-31含量分别上调116％ （P ＜0.05）和386％ （P ＜0.05）.与PDGF-BB组比较,PD98059＋PDGF-BB处理后,α-SMA含量显著上调41％ （P ＜0.05）,增殖型蛋白OPN和miR-31含量分别下调33％ （P ＜0.05）和46％（P＜0.05）.结论 阻断ERK通路可促进VSMCs由增殖型向收缩型转变,同时下调miR-31的表达水平,ERK-miR-31通路可能是调控人动脉硬化平滑肌细胞功能及表型的重要通路.

Objective To explore the effect of extracellular signal-regulated kinase （ERK）,microRNA （miR）-31 and platelet derived growth factor （PDGF）-BB interaction on the proliferation of smooth muscle cells.Methods Lower extremity amputation from patients diagnosed as having ASO in Guangdong School of Medicine Affiliated Hospital was used for primary culture of smooth muscle cells.Cell counting kit-8 （CCK-8）,Transwell,Western blotting,and real-time quantitative polymerase chain reaction （Real-time PCR） were used for evaluating PDGF-BB on the proliferation,migration and miR-31 expression on human VSMC cells.Results The tissue adherent method was used to successfully culture the original generation of arteriosclerosis smooth muscle cells.PDGF-BB enhanced proliferation rate of arteriosclerosis smooth muscle cells by 102% （P 〈 0.05）,and migration rate by 246% （P 〈 0.05）.PD98059 could block the effect of PDGF-BB,increasing the proliferation rate of smooth muscle cells by 53% （P 〈 0.05）,and migration rate by 19% （P 〈 0.05）.PDGF-BB could reduce shrinkage phenotypic marker protein α-smooth muscle actin （α-SMA） by 38% （P 〈 0.05）,but raise proliferative phenotypic marker protein of osteopontin （OPN） by 116% （P 〈 0.05） and miR-31 by 386% （P 〈 0.05）.As compared with PDGF-BB group,treatment of PD98059 ＋ PDGF-BB could significantly increase the α-SMA by 41% （P 〈0.05）,but decrease the proliferative protein of OPN and miR-31 by 33 % （P 〈 0.05） and 46% respectively （P 〈 0.05）.Conclusion Blocking the ERK pathway can change vascular smooth muscule cells from proliferative to shrinkage phenotypic,and down-regulate miR-31 expression level at the same time.ERK-miR-31 pathway may be one of important pathways in regulating phenotype and function of human arteriosclerotic smooth muscle cells.