中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2009年
3期
151-154,插1
,共5页
廖品琥%杜学柯%黄冰%姚为学%曹建
廖品琥%杜學柯%黃冰%姚為學%曹建
료품호%두학가%황빙%요위학%조건
细胞间黏附分子-1%核转录因子-κB%白细胞介素-1β
細胞間黏附分子-1%覈轉錄因子-κB%白細胞介素-1β
세포간점부분자-1%핵전록인자-κB%백세포개소-1β
intercellular adhesion molecule-1%nuclear factor-κB%interleukin-1β
目的 研究白细胞介素-1β(IL-1β)对A549细胞表达细胞间黏附分子-1(ICAM-1)的诱导作用及相关细胞内信号通路的激活和转导机制.方法 以1 μg/L终浓度的IL-1β刺激经SC-514[核转录因子-κB(NF-κB)的IKK-2复合物抑制物]预处理的A549细胞,用蛋白质免疫印迹法(Western blotting)检测IL-1β刺激5、10、30、60 min时细胞内磷酸化NF-κB抑制蛋白(pIκBα)和IκBα蛋白表达水平;激光扫描共焦显微镜(LSCM)显像检测NF-κB p65的核转移过程;按试剂盒说明测定NF-κB DNA结合活性;p65抗体染色质免疫沉淀结合聚合酶链反应(ChIP-PCR)技术检测乙酰化组蛋白H4和p65与ICAM-1基因启动子的结合;逆转录-聚合酶链反应(RT-PCR)检测4 h后的ICAM-1 mRNA表达;酶联免疫吸附法(ELISA)检测24 h后细胞表面的ICAM-1蛋白表达.结果 IL-1β刺激后细胞内pIκBα蛋白表达水平明显升高,IκBα蛋白表达水平明显下降;LSCM检测显示激活的p65蛋白从胞质向胞核转移,p65与DNA结合活性明显升高(P<0.01).ChIP-PCR扩增发现,IL-1β促使乙酰化组蛋白H4和p65与ICAM-1基因启动子区域的DNA片断结合.IL-1β刺激4 h后ICAM-1 mRNA表达明显升高,刺激24 h后A549细胞表面ICAM-1蛋白表达亦明显升高(P均<0.01).SC-514阻断了pIκBα蛋白的升高和IκBα蛋白的下降;减少了p65蛋白的核转移和DNA结合活性;降低了ICAM-1的mRNA及蛋白表达水平(P均<0.01).结论 IL-1β通过激活NF-κB 介导了A549细胞表达ICAM-1.
目的 研究白細胞介素-1β(IL-1β)對A549細胞錶達細胞間黏附分子-1(ICAM-1)的誘導作用及相關細胞內信號通路的激活和轉導機製.方法 以1 μg/L終濃度的IL-1β刺激經SC-514[覈轉錄因子-κB(NF-κB)的IKK-2複閤物抑製物]預處理的A549細胞,用蛋白質免疫印跡法(Western blotting)檢測IL-1β刺激5、10、30、60 min時細胞內燐痠化NF-κB抑製蛋白(pIκBα)和IκBα蛋白錶達水平;激光掃描共焦顯微鏡(LSCM)顯像檢測NF-κB p65的覈轉移過程;按試劑盒說明測定NF-κB DNA結閤活性;p65抗體染色質免疫沉澱結閤聚閤酶鏈反應(ChIP-PCR)技術檢測乙酰化組蛋白H4和p65與ICAM-1基因啟動子的結閤;逆轉錄-聚閤酶鏈反應(RT-PCR)檢測4 h後的ICAM-1 mRNA錶達;酶聯免疫吸附法(ELISA)檢測24 h後細胞錶麵的ICAM-1蛋白錶達.結果 IL-1β刺激後細胞內pIκBα蛋白錶達水平明顯升高,IκBα蛋白錶達水平明顯下降;LSCM檢測顯示激活的p65蛋白從胞質嚮胞覈轉移,p65與DNA結閤活性明顯升高(P<0.01).ChIP-PCR擴增髮現,IL-1β促使乙酰化組蛋白H4和p65與ICAM-1基因啟動子區域的DNA片斷結閤.IL-1β刺激4 h後ICAM-1 mRNA錶達明顯升高,刺激24 h後A549細胞錶麵ICAM-1蛋白錶達亦明顯升高(P均<0.01).SC-514阻斷瞭pIκBα蛋白的升高和IκBα蛋白的下降;減少瞭p65蛋白的覈轉移和DNA結閤活性;降低瞭ICAM-1的mRNA及蛋白錶達水平(P均<0.01).結論 IL-1β通過激活NF-κB 介導瞭A549細胞錶達ICAM-1.
목적 연구백세포개소-1β(IL-1β)대A549세포표체세포간점부분자-1(ICAM-1)적유도작용급상관세포내신호통로적격활화전도궤제.방법 이1 μg/L종농도적IL-1β자격경SC-514[핵전록인자-κB(NF-κB)적IKK-2복합물억제물]예처리적A549세포,용단백질면역인적법(Western blotting)검측IL-1β자격5、10、30、60 min시세포내린산화NF-κB억제단백(pIκBα)화IκBα단백표체수평;격광소묘공초현미경(LSCM)현상검측NF-κB p65적핵전이과정;안시제합설명측정NF-κB DNA결합활성;p65항체염색질면역침정결합취합매련반응(ChIP-PCR)기술검측을선화조단백H4화p65여ICAM-1기인계동자적결합;역전록-취합매련반응(RT-PCR)검측4 h후적ICAM-1 mRNA표체;매련면역흡부법(ELISA)검측24 h후세포표면적ICAM-1단백표체.결과 IL-1β자격후세포내pIκBα단백표체수평명현승고,IκBα단백표체수평명현하강;LSCM검측현시격활적p65단백종포질향포핵전이,p65여DNA결합활성명현승고(P<0.01).ChIP-PCR확증발현,IL-1β촉사을선화조단백H4화p65여ICAM-1기인계동자구역적DNA편단결합.IL-1β자격4 h후ICAM-1 mRNA표체명현승고,자격24 h후A549세포표면ICAM-1단백표체역명현승고(P균<0.01).SC-514조단료pIκBα단백적승고화IκBα단백적하강;감소료p65단백적핵전이화DNA결합활성;강저료ICAM-1적mRNA급단백표체수평(P균<0.01).결론 IL-1β통과격활NF-κB 개도료A549세포표체ICAM-1.
Objective To investigate the effects of interleukin-1β (IL-1β) on intercellular adhesion molecule-1 (ICAM-1) expression on A549 cell and underlying signal transduction pathways. Methods A549 cells were pre-incubated with SC-514 [nuclear factor-κB (NF-κB) inhibitor (IκB) kinase-2 (IKK-2) inhibitor] and/or pre-treated with 1 μg/L IL-1β. The phosphorylated IκBα (pIκBα) and degradation of IκBα were determined by Western blotting with specific antibody at 5, 10, 30 and 60 minutes. Laser scanning confocal microscope (LSCM) was used to examine the nuclear translocation of p65 at 30 minutes after stimulation. The DNA binding activity of p65 in nuclear extracts was detected at 1 hour following IL-1β treatment. Chromatin immunoprecipitation (ChIP) assays combined with polymerase chain reaction (PCR) were used to evaluate interaction between p65 and ICAM-1 promoter site DNA at 1 hour after stimulation. The expression of ICAM-1 mRNA was assessed by reverse transcription (RT)-PCR at 4 hours, and the ICAM-1 expression on A549 cell surface was measured by enzyme linked immunosorbent assay (ELISA) at 24 hours after IL-1β was added. Results IL-1β induced rapid pIκBα augmentation and its subsequent degradation. LSCM graphs showed that IL-1β stimulated the translocation of p65 from the cytosol to the nucleus. IL-1β significantly increased the DNA binding ability of p65 (P<0.01) in cell nuclear extracts. ChIP-PCR suggested that both acetylated histone 4 and p65 were recruited to ICAM-1 promoter. IL-1β significantly augmented ICAM-1 mRNA level at 4 hours and expression of ICAM-1 on A549 cell surface at 24 hours (both P<0.01). The IKK-2 inhibitor, SC-514, inhibited IL-1β induced IκBα protein activity, blocked p65 nuclear translocation, caused a significant reduction in IL-1β induced DNA binding activity for p65 and ICAM-1 mRNA expression, and suppressed ICAM-1 expression on A549 cell surface (all P<0.01). Conclusion These results suggest that the activation of NF-κB mediates IL-1β induced ICAM-1 expression in A549 cells.
