西安交通大学学报(医学版)
西安交通大學學報(醫學版)
서안교통대학학보(의학판)
JOURNAL OF XI'AN JIAOTONG UNIVERSITY(MEDICAL SCIENCES)
2015年
3期
317-321
,共5页
杨旭东%宁启兰%蒋晓刚%孙青竹%刘莉%韩燕%张富军%王慧莲
楊旭東%寧啟蘭%蔣曉剛%孫青竹%劉莉%韓燕%張富軍%王慧蓮
양욱동%저계란%장효강%손청죽%류리%한연%장부군%왕혜련
哮喘%大鼠%上皮%IL-4%G 蛋白偶联的内整流钾通道
哮喘%大鼠%上皮%IL-4%G 蛋白偶聯的內整流鉀通道
효천%대서%상피%IL-4%G 단백우련적내정류갑통도
asthma%rat%epithelium%IL-4%G protein-coupled inwardly rectifying potassium channel
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14 d 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50 ng/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。
目的:檢測 G 蛋白偶聯的內整流鉀通道(G protein-coupled inwardly rectifying potassium channels,GIRK)在哮喘模型中的錶達改變,併尋找調節其錶達變化的因素。方法用卵清蛋白和鋁佐劑緻敏 E3大鼠14 d 後,用卵清蛋白進行攻擊以誘導哮喘模型,用燐痠鹽緩遲液緻敏併攻擊的大鼠作為對照,通過觀察肺組織病變、總 IgE 水平的改變等手段評價哮喘模型。用 RT-PCR 檢測 GIRK 亞基1~4的 mRNA 水平,用 Western blot 等 手 段 檢測 大 鼠 肺 組 織 中GIRK1和 GIRK3的蛋白水平,併用免疫組化確定肺組織中 GIRK 錶達的解剖部位。根據免疫組化的結果,選用A549細胞株作為模型,用 IL-4刺激,用 PCR、Western blot 等手段檢測 IL-4對 GIRK 錶達的影響。結果與對照組相比,哮喘模型組肺組織中 GIRK 的 mRNA 水平下降,哮喘模型組肺組織中 GIRK 蛋白水平也下降。免疫組化結果顯示,GIRK 在大鼠主要錶達于氣道上皮。隨 IL-4濃度的升高,在 A549細胞中 GIRK 的所有的亞基的錶達均呈劑量依賴性下降趨勢;選擇50 ng/mL IL-4刺 激 A549細 胞,GIRK 的所有的亞基的錶達均呈刺激時間依賴性的下降。結論在 E3大鼠哮喘模型中,IL-4下調氣道上皮中 GIRK 的錶達。
목적:검측 G 단백우련적내정류갑통도(G protein-coupled inwardly rectifying potassium channels,GIRK)재효천모형중적표체개변,병심조조절기표체변화적인소。방법용란청단백화려좌제치민 E3대서14 d 후,용란청단백진행공격이유도효천모형,용린산염완충액치민병공격적대서작위대조,통과관찰폐조직병변、총 IgE 수평적개변등수단평개효천모형。용 RT-PCR 검측 GIRK 아기1~4적 mRNA 수평,용 Western blot 등 수 단 검측 대 서 폐 조 직 중GIRK1화 GIRK3적단백수평,병용면역조화학정폐조직중 GIRK 표체적해부부위。근거면역조화적결과,선용A549세포주작위모형,용 IL-4자격,용 PCR、Western blot 등수단검측 IL-4대 GIRK 표체적영향。결과여대조조상비,효천모형조폐조직중 GIRK 적 mRNA 수평하강,효천모형조폐조직중 GIRK 단백수평야하강。면역조화결과현시,GIRK 재대서주요표체우기도상피。수 IL-4농도적승고,재 A549세포중 GIRK 적소유적아기적표체균정제량의뢰성하강추세;선택50 ng/mL IL-4자 격 A549세 포,GIRK 적소유적아기적표체균정자격시간의뢰성적하강。결론재 E3대서효천모형중,IL-4하조기도상피중 GIRK 적표체。
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.