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

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

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Western blot检测细胞磷酸化ERK2蛋白水平；噻唑蓝（MTF）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。

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 10μg/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 distributon 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 percentage and migrated cell number in Adanti-ERK2 group and control group were all significantly reduced as compared with those in PDGF group and Ad-LaeZ group （ all P 〈 0.05 ）. The supernatant TGF-β1 levels in Adanti-ERK2 group and control group were both significantly lower than those in PDGF group and AdLacZ group;the TGF-β1 levels in Adanti-ERK2 group were higher than in control group P 〈 0.05 ）. Conclusion 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 stimulated with PDGF-BB. ERK signaling pathway participates in PDGF-BB induced VSMCs proliferation, migration and gene expression.