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目的探讨Elafin在胞质内对表皮生长因子(EGF)诱导的黏蛋白(MUC)5AC生成的影响及其作用机制。方法体外培养人支气管上皮细胞系16HBE,将已构建好的pEGFP-N1-Elafin载体通过LipofectamineTM2000转染到16HBE细胞中,以转染空质粒载体的16HBE细胞作为对照,均给予外源性EGF作为刺激物共同孵育。以RT-PCR和ELISA分别检测细胞内黏蛋白(MUC)5AC mRNA水平和MUC5AC含量。间接免疫荧光细胞化学法结合激光共聚焦显微镜检测细胞核中核因子(NF)-κB的活性。免疫共沉淀法结合Western blot检测SUMO-1-p-IκBα含量。结果转染重组质粒的细胞中MUC5ACmRNA水平、MUC5AC的含量(0.36±0.09)以及NF-κB的活性显著低于转染空载体的对照组细胞(MUC5AC的含量0.55±0.07)(P<0.01),而SUMO-1-p-IκBα的含量则显著高于后者(P<0.01)。结论 Elafin能够通过促进p-IκBα的类泛素化阻止NF-κB的活化,从而下调MUC5AC的生成。
Objective To investigate the effect of Elafin on the production of epidermal growth factor (EGF) -mediated mucin (MUC) 5AC in the cytoplasm and its mechanism. Methods Human bronchial epithelial cell line 16HBE was cultured in vitro. The constructed pEGFP-N1-Elafin vector was transfected into 16HBE cells by LipofectamineTM2000. The 16HBE cells transfected with empty plasmid vector served as controls, and exogenous EGF was given as stimulus Co-incubation. The levels of intracellular mucin (MUC) 5AC mRNA and MUC5AC were detected by RT-PCR and ELISA, respectively. Indirect immunofluorescence cytochemistry combined with confocal laser scanning microscopy to detect nuclear factor (NF) -κB activity. Immunoprecipitation and Western blot were used to detect the expression of SUMO-1-p-IκBα. Results The MUC5AC mRNA level, MUC5AC level (0.36 ± 0.09) and NF-κB activity in transfected cells were significantly lower than those in control cells transfected with empty vector (MUC5AC 0.55 ± 0.07) (P <0.01) While the content of SUMO-1-p-IκBα was significantly higher than the latter (P <0.01). Conclusion Elafin can down-regulate the production of MUC5AC by blocking ubiquitination of p-IκBα and blocking the activation of NF-κB.