G蛋白偶联的内整流型钾通道在哮喘模型大鼠肺组织的表达

The expression of G protein-coupled inwardly rectifying potassium channels in rat asthma model

目的检测G蛋白偶联的内整流钾通道（G protein-coupled inwardly rectifying potassium channels,GIRK）在哮喘模型中的表达改变,并寻找调节其表达变化的因素。方法用卵清蛋白和铝佐剂致敏E3大鼠14d后,用卵清蛋白进行攻击以诱导哮喘模型,用磷酸盐缓冲液致敏并攻击的大鼠作为对照,通过观察肺组织病变、总IgE水平的改变等手段评价哮喘模型。用RT-PCR检测GIRK亚基1-4的mRNA水平,用Western blot等手段检测大鼠肺组织中GIRK1和GIRK3的蛋白水平,并用免疫组化确定肺组织中GIRK表达的解剖部位。根据免疫组化的结果,选用A549细胞株作为模型,用IL-4刺激,用PCR、Western blot等手段检测IL-4对GIRK表达的影响。结果与对照组相比,哮喘模型组肺组织中GIRK的mRNA水平下降,哮喘模型组肺组织中GIRK蛋白水平也下降。免疫组化结果显示,GIRK在大鼠主要表达于气道上皮。随IL-4浓度的升高,在A549细胞中GIRK的所有的亚基的表达均呈剂量依赖性下降趋势;选择50ng/mL IL-4刺激A549细胞,GIRK的所有的亚基的表达均呈刺激时间依赖性的下降。结论在E3大鼠哮喘模型中,IL-4下调气道上皮中GIRK的表达。

Objective To detect the changes of G protein-coupled inwardly rectifying potassium channels （GIRK）expression in allergic asthma model and identify the regulatory factors.Methods The E3 rat asthma models were induced by challenge with ovalbumin 14 days after immunization with ovalbumin and aluminium adjuvant.The asthma models were evaluated based on changes in lung pathomorphology and total IgE levels.The levels of GIRK1-4 mRNA and protein were detected using real time-PCR and Western blot.The anatomic sites where GIRK was expressed dominantly in the lung were identified using immunohistological staining.To identify the effects of IL-4 on the expressions of GIRK channels,GIRK 1 -4 mRNA and protein in IL-4 stimulated bronchial epithelial cell line A549 were detected by RT-PCR and Western blot.Results The levels of GIRK1-4 mRNA and protein decreased significantly in the lung in asthmatic E3 rats.The results of immunohistological staining showed that GIRK channels were dominantly expressed in airway epithelia in the lung.The levels of GIRK 1-4 mRNA and protein were down-regulated in time-and dose-dependent manners in IL-4 treated A549.Conclusion IL-4 down-regulates the expression levels of GIRK subunits in bronchial epithelia during allergic asthma.