ERK活化对哮喘大鼠气道重塑及CyclinD1表达的影响

目的探讨细胞外信号调节蛋白激酶（ERK）对哮喘大鼠气道重塑及CyclinD1表达的作用。方法原代培养大鼠的平滑肌细胞（ASMCs）,给予ERK激动剂表皮生长因子EGF和抑制剂PD98059干预ASMCs生长,依处理方式不同分为5组：（1）正常对照组（2）哮喘对照组;（3）E组：EGF20 ng/mL;（4）P＋E组,PD98059 10μmol/L1 h后添加EGF 20 ng/mL;（5）PD组,PD98059 10μmol/L。采用四甲基偶氮唑盐（MTT）法检测气道平滑肌细胞（ASMCs）增殖能力,流式细胞术（FCM）测定细胞周期和cyclinD1的蛋白含量,RT-PCR方法检测cyclinD1mRNA表达水平。结果（1）与哮喘对照组比较,E组ASMCs S＋G2/M期比例、吸光度A值、cyclinD1蛋白阳性表达率和cyclinD1 mRNA的A值均显著升高,PD组均显著降低（P〈0.05）。P＋E组与哮喘对照在此4项指标上比较无明显差异。（2）哮喘（对照组、E组、PD组和P＋E组）组与正常对照组,其S＋G2/M期比例、吸光度A值、cyclinD1蛋白和cyclinD1 mRNA的表达均显著增高（P〈0.05）。结论ERK活性促进哮喘大鼠ASMCs的增殖,增加cyclinD1在哮喘平滑肌细胞中的表达,导致气道重塑的形成,提示ERK可能对CyclinD1的表达具有调节作用。

Effect of ERK on Asthmatic Rat Airway Remodeling and CyclinD1

Objective To investigate the effects of ERK on asthma airway remodeling and the expressions of cyclinD1.Methods The rat asthma model was made through ovalbumin（OVA） sensitization and excitation.Primary cultures of ASMCs were established and cells between passages 3 and 6 were used for experiments.ASMCs were treated with ERK activator epidermal growth factor（EGF） and inhibitor PD98059,and then subdivided into 5 groups based on the drug interventions：（1）Normal control group;（3）Asthmatic control group;（3）Asthmatic EGF group;（4）P＋E group,PD98059 10 μmol/L ＋EGF 20 ng/mL;（5） PD98059 group.The proliferations of ASMCs were examined with cell cycle analysis and MTT colorimetric assay,the expressions of cyclinD1 mRNA were detected by RT-PCR,and the protein expression of cyclinD1 was measured by FCM.Results Compared to asthmatic control group,the percentage of cells at S＋G_2/M phase,A value,the expression of cyclinD1 mRNA and cyclinD1 protein from E group were significantly increased（P0.05）;the PD group were significantly reduced respectively（P0.05）.There was no significant difference between P＋ E group and the asthmatic control group in the above four index.（2）Compared with normal control group,the percentage of cells at S＋G_2/M phase,A value,the expression of cyclinD1 mRNA and cyclinD1 protein from asthmatic group（control group,E group,P group and P＋E group） were significantly increased（P0.05）.Conclusion The activation of ERK promotes ASMCs proliferation and expressions of cyclinD1 in asthmatic rats,indicating that the expression of CyclinD1 may be dependent on the ERK activation in asthmatic rats.The finding will help to understand the mechanisms of airway remodeling.