摘要
目的探讨TNF-α对哮喘大鼠气道平滑肌细胞(ASMCs)增殖及对ASMCs上ERK1/2 mRNA、p-ERK1/2表达水平的影响。方法通过对哮喘模型大鼠ASMCs培养,分别以0.2μg/L、1.0μg/L、20μg/L TNF-α干预ASMCs生长。采用流式细胞仪、MTT法检测ASMCs增殖情况,观察不同浓度TNF-α对ASMCs增殖的影响。RT-PCR检测ASMCs上ERK1/2 mRNA表达,免疫细胞化学染色法检测磷酸化ERK1/2蛋白的表达及定位。结果哮喘组ASMCsS期比例、A值、ERK1/2 mRNA、p-ERK1/2蛋白的表达量分别为(34.45±2.08)%、(0.550±0.010)、(0.995±0.118)、(130.77±4.16),与对照组(11.17±0.96)%、(0.292±0.008)、(0.576±0.098)、(163.82±1.38)比较均显著增高(均P<0.01)。各TNF-α干预组ASMCs的S期比例、A值、ERK1/2 mRNA和p-ERK1/2蛋白表达量与哮喘组比较均显著降低(均P<0.01),0.2μg/L和1.0μg/L TNF-α组p-ERK1/2蛋白表达量高于对照组(P<0.01),20μg/L TNF-α组p-ERK1/2蛋白表达量与对照组比较无差异(P>0.05)。结论与正常鼠相比,慢性哮喘大鼠气道平滑肌细胞增殖明显,处于S期的细胞比例明显增高。经TNF-α干预后,慢性哮喘大鼠气道平滑肌细胞处于S期的细胞比例减少,增殖减弱,TNF-α可能抑制慢性哮喘大鼠气道平滑肌细胞增殖。TNF-α可下调慢性哮喘大鼠气道平滑肌细胞上ERK1/2 mRNA及p-ERK1/2表达,TNF-α可能通过抑制ERK信号转导通道的活性对气道平滑肌细胞的增殖进行调控。
Objective To investigate the effect of TNF-α on airway smooth muscle cells （ASMCs） proliferation and expression of ERK1/2 mRNA and P-ERK1/2 in asthmatic rats. Methods The rat asthma model was made through ovalbumin （OVA） sensitization and excitation. Primary cultures of ASMCs were established for experiments. ASMCs were treated with 0.2 μg/L, 1.0μg/L, 20 μg/L TNF-α. Proliferation of ASMCs was detected by flow cytometry and MTT colorimetric assay, observation of different concentrations of TNF-α on the proliferation of ASMCs. The expressions of ERK1/2 mRNA were detected by RT-PCR, the location and protein expressions of phosphorylated forms of ERK1/2 （ p-ERK1/2 ） were examined by immunocytochemical staining. Results The percentage of cells at S phase, A value, The expression quantities of ERK mRNA and p-ERK1/2 protein from asthmatic group were （34.45 ±2.08）%, （0.550 ± 0.010）, （0.995 ± 0.118）, （130.77± 4.16）respectively, significantly increased compared with the control group[ （11.17 ± 0.96）%, （0.292 ± 0.008）, （0.576 ± 0.098）, （163.82 ± 1.38）respectively, （P 〈 0.01）1; ASMCs in asthmatic group were intervened by the different concentrations of TNF-α, the percentage of cells at S phase, A value, the expression quantities of ERK mRNA and p-ERK1/2 protein were significantly lower than those in the asthmatic group（ P 〈 0.01 ）. However, the expression quantities Of p-ERK1/2 protein in ASMCs from 0.2μg/L and 1.0 μg/L TNF-α group were higher than that from control group（ P 〈 0.01 ）, the expression quantities of p-ERK1/2 protein from 20 μg/L TNF-α group were not different from those from control group （ P 〉 0.05 ）. Conclusion Compared with normal rats, the proliferation of ASMCs in chronic asthmatic rats was obvious, the percentage of cells at S phase was increasing. However, ASMCs in chronic asthmatic rats was intervened by TNF-α, the percentage of cells at S phase was decreased and the proliferation of ASMCs was reduced, TNF-α may inhibit the proliferation of ASMCs in chronic asthmatic rats. TNF-α down-regulated Expression of ERK1/2 mRNA and P-ERK1/2 in asthmatic rats, TNF-α may regulate the proliferation of ASMCs in chronic asthmatic rats by inhibiting the activity of ERK signaling pathway.
出处
《中国比较医学杂志》
CAS
2009年第7期23-28,I0006,共7页
Chinese Journal of Comparative Medicine
基金
山西省教育厅2005年山西省高校科技研究开发项目(NO.20051001)
关键词
肿瘤坏死因子-Α
细胞外信号调节激酶
气道平滑肌细胞
增殖
Tumor necrosis factor-alpha （TNF-α）
Extracellular signal-regulated kinase （ERK）
Airway smooth muscle cells（ASMCs）
Proliferation
