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目的探讨天然内生多肽Elafin对脂多糖诱导的气道黏液高分泌的调节机制。方法构建人Elafin重组质粒pEGFP-N1-Elafin,转染正常人支气管上皮细胞HBE16,给予脂多糖(LPS)刺激,激光共聚焦扫描显微镜检测Elafin蛋白含量,Western blot法检测IκBα蛋白含量;荧光素酶报告基因检测系统测定核转录因子-κB(NF-κB)活性;RT-PCR检测Elafin和黏蛋白(MUC)5AC mRNA表达水平;ELISA法分析MUC5AC蛋白相对含量。结果成功构建内生多肽Elafin真核表达载体pEGFP-N1-Elafin并转染HBE16细胞。转染重组Elafin后Elafin蛋白含量及mRNA水平明显增加。与对照组相比,LPS刺激组IκBα蛋白含量降低,NF-κB相对活性明显增强,MUC5AC蛋白含量及mRNA水平也显著增加。转染重组Elafin后再予以LPS刺激,与单纯LPS刺激组相比,IκBα蛋白含量明显增加,NF-κB相对活性显著降低,MUC5AC蛋白含量及mRNA水平也明显降低。结论天然内生多肽Elafin重组气道上皮可下调NF-κB的活性,降低脂多糖诱导的气道黏液高分泌。
Objective To investigate the regulatory mechanism of natural endogenous polypeptide Elafin on airway mucus hypersecretion induced by lipopolysaccharide. Methods Human Elafin recombinant plasmid pEGFP-N1-Elafin was transfected into normal human bronchial epithelial cells HBE16 and stimulated by lipopolysaccharide (LPS). The content of Elafin protein was detected by confocal laser scanning microscopy. The content of IκBα protein was detected by Western blot. The luciferase The reporter gene detection system was used to determine the activity of nuclear transcription factor-κB (NF-κB). The expression of Elafin and MUC 5AC mRNA was detected by RT-PCR and the relative content of MUC5AC protein by ELISA. Results The Elafin eukaryotic expression vector pEGFP-N1-Elafin was successfully constructed and transfected into HBE16 cells. Elafin protein content and mRNA levels were significantly increased after transfection of recombinant Elafin. Compared with the control group, the content of IκBα protein decreased, the relative activity of NF-κB increased significantly, and the content of MUC5AC protein and mRNA level increased significantly in LPS stimulation group. Transfection of recombinant Elafin followed by LPS stimulation, compared with LPS stimulation group, IκBα protein content was significantly increased, NF-κB relative activity was significantly reduced, MUC5AC protein content and mRNA levels were significantly reduced. Conclusion The natural endogenous peptide Elafin recombinant airway epithelium can down-regulate the activity of NF-κB and decrease the secretion of airway mucus induced by lipopolysaccharide.