冷刺激诱导哮喘小鼠支气管上皮细胞炎症反应的研究

Study on inflammatory response of bronchial epithelial cells induced by cold stimulation in asthmatic mice

目的探讨冷刺激对哮喘小鼠支气管上皮细胞炎症反应的影响以及蛋白激酶C(PKC)-核因子-κB(NF-κB)信号转导通道的变化情况。方法原代分离培养哮喘模型小鼠支气管上皮细胞,并将细胞分为对照组(37℃培养)、24 h冷刺激组(18℃培养)和48 h冷刺激组(18℃培养),实时荧光定量PCR方法检测三组细胞炎性细胞因子白细胞介素(IL)-1α、IL-1β、IL-4、IL-6、IL-8、IL-10、IL-13、粒细胞-巨噬细胞集落刺激因子(GM-CSF)及肿瘤坏死因子(TNF)-αmRNA表达,免疫荧光法检测磷酸化PKC及磷酸化NF-κB抑制因子(IκB)并测定其荧光值以判定PKC及NF-κB活性。结果各冷刺激后,除IL-1αmRNA表达量太低无法进行分析外,其余各基因表达量均显著升高(P<0.05)。其中IL-1β和GM-CSF表达在冷刺激24 h后增加最为明显,IL-13表达在冷刺激48 h后增加最为明显,而IL-4、IL-6、IL-8、IL-10及TNF-α冷刺激24 h和48 h,作用效果基本一致。并且与对照组相比,24 h冷刺激组和48 h冷刺激组的磷酸化PKC、磷酸化IκB蛋白表达量均明显增加(P<0.05)。结论冷刺激能够诱导哮喘小鼠支气管上皮细胞的炎症反应,而PKC-NF-κB可能是这一过程中的重要信号转导途径。

Objective To explore the effect of cold stimulation on inflammatory response of bronchial epithelial cells in asthmatic mice and the changes in protein kinase C （PKC） - nuclear factor - κB （ NF - κB ） signal transduction pathway. Methods The cultured bronchial epithelial cells of asthmatic mice were divided into control group （cultured at 37℃） , 24 h cold stimulation group （ cultured at 18） and 48 h cold stimulation group （ cultured at 18℃ ）. The expression levels of inflammatory cell factors as interleukin （IL） -1α〉IL-1β,IL-4,IL-6,IL -8 , IL - 10 , IL - 13 , granulocyte - macrophage colony stimulating factor （ GM - CSF） and tumor necrosis factor （TNF - a ） mRNA were detected by real time quantitative PCR, phosphorylated PKC and phosphorylated NF - κB inhibitor （ IκB） were detected by fluoroimmunoassay, and the fluorescent values were detected to determine the activity of PKC and NF - κB. Results After cold stimulation, the expression level of IL - α gene was too low to be analyzed, the expression levels of other genes were significantly increased （ P 〈0. 0 5） . The expression of IL - 1β and GM -CSF in 24 h after cold stimulation was significantly increased, the expression level of IL - 13 in 48 h after cold stimulation was significantly in-creased ,while the expression levels of IL - 4, IL - 6, IL - 8 , IL - 10 and TNF -α in 24 h and 48 h after cold stimulation were basically the same. In comparison with the control group, expression levels of phosphorylated PKC and phosphorylated IκB protein were significantly increased in 24 h and 48 h after cold stimulation （ P 〈 0. 0 5） . Conclusion Cold stimulation induces the inflammatory response of asthmatic bronchial epithelial cells, and PKC and NF - κB are important signal transduction molecules in signal pathway.