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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。
This article aims to investigate the role of extracellular signal-regulated kinase (ERK) in the proliferation of airwaysmooth muscle cells (ASMCs) in chronic bronchial asthma rats. A rat model of chronic asthma was established. The ERK agonist epidermal growth factor (EGF) and inhibitor PD98059 were used to induce the culture of ASMCs in chronic asthmatic rats. The proliferation of ASMCs was detected by flow cytometry, MTT assay, 3H-thymidine (TdR) incorporation and proliferating cell nuclear antigen (PCNA) immunohistochemistry. The expression of ERK Effects of Signal Pathway on Proliferation of ASMCs. The expression of ERK mRNA and ERK1 / 2, phosphorylated ERK1 / 2 (p-ERK1 / 2) protein were detected by RT-PCR and Western blot. Compared with normal control group, the percentage of G0 / G1 phase cells in ASMCs in chronic asthma group was significantly decreased, and the proportion of cells in S + G2 / M phase increased. The absorbance (A490), DNA synthesis and PCNA positive expression ERK mRNA, ERK1 / 2 protein, p-ERK1 / 2 protein expression and ERK activation were significantly increased. After intervention with PD98059, the proportion of cells in S + G2 / M phase, A490 value, the amount of DNA synthesis and the expression of PCNA in ASMCs of chronic asthmatic group were significantly decreased. The expressions of ERK mRNA, ERK1 / 2 protein and p-ERK1 / 2 protein The amount of expression and ERK activation rate was significantly reduced. After EGF intervention, the percentage of S + G2 / M phase cells, A490 value, DNA synthesis and PCNA expression in ASMCs of chronic asthma group were further increased, and this effect was inhibited by PD98059. The above results suggest that endogenous proliferation of ASMCs in chronic asthmatic rats is increased, and ERK1 / 2 is involved in the regulation of proliferative activity. ERK signaling plays an important role in the proliferation of asthmatic ASMCs.