摘要
本文旨在探讨细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)在慢性支气管哮喘大鼠气道平滑肌细胞(airwaysmooth muscle cells, ASMCs)增殖中的作用。建立慢性哮喘大鼠模型,用 ERK 激动剂表皮生长因子(epidermal growth factor,EGF)和抑制剂 PD98059 干预慢性哮喘大鼠 ASMCs 的培养。采用流式细胞仪、四甲基偶氮唑盐(MTT)法、3H-thymidine (TdR)掺入法和增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)免疫组织化学法检测ASMCs增殖情况,观察ERK信号通路对ASMCs 增殖的影响。RT-PCR 和 Western blot 检测 ERK mRNA 和 ERK1/2、磷酸化 ERK1/2 (p-ERK1/2)蛋白的表达。与正常对照组ASMCs 比较,慢性哮喘组ASMCs 的 G0/G1 期细胞所占比例明显减少,S+G2/M 期细胞所占比例增高;吸光度(A490)值、细胞DNA合成量和PCNA阳性表达量均明显增加,ERK mRNA、ERK1/2 蛋白、p-ERK1/2 蛋白的表达量以及ERK 活化率显著增高。经PD98059 干预之后,慢性哮喘组ASMCs 的 S+G2/M 期细胞所占比例、A490 值、细胞DNA合成量和PCNA阳性表达量明显降低,ERK mRNA、ERK1/2 蛋白、p-ERK1/2 蛋白的表达量以及ERK 活化率显著降低。经EGF 干预后,慢性哮喘组ASMCs 的 S+G2/M 期细胞所占比例、A490 值、细胞DNA合成量和PCNA阳性表达量进一步增高,而这一作用可以被PD98059 抑制。以上结果提示,慢性哮喘大鼠ASMCs 内源性增殖活性增加,ERK1/2 参与其增殖活性的调控,ERK 信号通路在哮喘气道重建的ASMCs 增殖调控中具有重要作用。
To investigate the regulatory effect of extracellular signal-regulated kinase (ERK) signaling pathway on airway smooth muscle cell (ASMC) proliferation in chronic asthmatic rats, the rat model of chronic asthma was established, and ERK agonist epidermal growth factor (EGF) and inhibitor PD98059 were used in the cell culture. ASMC proliferation was examined by flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [^3H]-thymidine (TdR) incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of ERK mRNA, ERK protein, phosphorylated ERK1/2 (p-ERK1/2) protein were observed by RT-PCR and Western blot. The results showed that in chronic asthmatic group, compared with that in the control group, the percentage of cells at G0/G1 phase was significantly decreased and the percentage of cells at S+G2/M phase was significantly increased. Absorbance (A490), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased. The expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly increased compared with those in the control group. After treatment with PD98059, the percentage of cells at S+G2/M phase, A490, DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly decreased; the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly decreased compared with those in the control group. After treatment with EGF, the percentage of cells at S+G2/M phase, A490, DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased compared with those before treatment; and PD98059 markedly inhibited the effect of EGF. These results suggest that the endogenous proliferation activity of ASMCs in chronic asthmatic rats significantly increases compared with that in the control rats, and ERK1/2 participates in this process. The ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.
出处
《生理学报》
CAS
CSCD
北大核心
2007年第3期311-318,共8页
Acta Physiologica Sinica
基金
the National Natural Science Foundation of China (No. 30400195)
grants from the Youth ChenguangScience Project of Wuhan Municipality (No. 196024047)
关键词
哮喘
细胞外信号调节激酶
平滑肌细胞
增殖
asthma
extracellular signal-regulated kinase
smooth muscle cell
proliferation