阻断ERK2信号转导通路对血小板源生长因子-BB诱导血管平滑肌细胞增殖、迁移和表达转化生长因子-β1的影响

目的 观察ERK2信号转导通路在血小板源生长因子(PDGF-BB)诱导血管平滑肌细胞(VSMC)增殖、迁移和表达转化生长因子(TGF)-β1中的作用.方法 将原代培养的大鼠VSMC分为4组:(1)对照组;(2)PDGF刺激组;(3)Ad-LacZ组;(4)Adanti-ERK2组.用Westernblot检测细胞磷酸化ERK2蛋白水平;噻唑蓝(MTT)比色法测定细胞增殖率;流式细胞仪检测细胞细胞周期;Boyden小室测定细胞的跨膜迁移能力;酶联免疫吸附试验(ELISA)法检测细胞培养液上清中TGF-β1的浓度.结果 Adanti-ERK2组和对照组细胞增殖率、S期细胞百分比及跨膜迁移细胞数目明显低于PDGF刺激组和Ad-LacZ组(细胞增殖率:2.75%、0.00%比64.45%、61.88%;s期细胞百分比:14.18%、13.58%比38.14%、32.99%;跨膜迁移细胞数:8.2±3.2、6.3±2.6比24.8±6.1、23.3±5.8,均P<0.05);(2)Adanti-ERK2组和对照组细胞培养上清液中TGF-β1含量明显低于PDGF刺激组和Ad-LacZ组(P<0.05);而Adanti-ERK2组明显高于对照组(P<0.05).结论 ERK2信号转导通路参与调控PDGF-BB诱导的VSMC增殖、迁移和基因表达.反义ERK2基因预先转染阻断ERK信号转导能显著抑制PDGF-BB刺激的VSMC增殖、迁移,阻断细胞周期由G1期进入S期,并且部分下调TGF-β1的合成分泌.

Impact of ERK2 signaling pathway blockade on platelet-derived growth factor-induced vascular smooth muscle cells proliferation,migration and transform growth factar-β1 expression

Objective To investigate the effects of ERK pathway on the proliferation, migration and TGF-β1 expression of vascular smooth muscle cells (VSMC) induced by platelet-derived growth factor (PDGF-BB). Methods Primary cultured rat VSMCs were divided into control group, PDGF-BB group (stimulated by 10 μg/L PDGF-BB), Ad-LacZ group (intervened by Ad-LacZ + PDGF-BB) and Adanti-ERK2 group (intervened by Adanti-ERK2 + PDGF-BB). The phosphorylated protein level of ERK2 was determined by Western blot analysis. The proliferative effect of the cells was evaluated by MTT. The dis-tributon of cell cycle was detected by flow cytometry. The cell migration was measured by the Boyden chamber. The TGF-β1 expression was assayed by ELISA. Results The proliferative rate,S stage percent-age and migrated ceil number in Adanti-ERK2 group and control group were all significantly reduced as compared with those in PDGF group and Ad-LacZ group (all P < 0.05). The supematant TGF-β1 levels in Adanti-ERK2 group and control group were both significantly lower than those in PDGF group and Ad-LacZ group;the TGF-β1 levels in Adanti-ERK2 group were higher than in control group P <0.05). Con-clusion The blockade of ERK signaling pathway by Adanti-ERK2 dominantly inhibited the proliferation and migration,arrested cells in G1 phase and partially down-regulated TGF-β1 expression in VSMCs stimu-lated with PDGF-BB. ERK signaling pathway participates in PDGF-BB induced VSMCs proliferation, mi-gration and gene expression.