摘要目的 探索不分型流感嗜血杆菌(NTHi)上调人类MUC5AC黏液素基因表达的细胞分子机制.方法 通过反转录套式-PCR及实时荧光定量PCR分析NTHi所诱导的MUC5AC mRNA的表达,免疫沉淀及Western印迹法检测NTHi对表皮生长因子受体(EGFR)及p38丝裂原活化蛋白激酶(p38MAPK)的激活,采用p38 MAPK或EGFR特异性抑制剂,共转染EGFR显性失活质粒,判断在转录水平对NTHi所诱导的MUC5AC表达的影响,并研究EGFR特异性抑制剂对NTHi所诱导p38MAPK激活的影响.结果 在人结肠上皮细胞株(HM3)细胞中,NTHi在mRNA及转录水平上调人类MUC5AC黏液素基因的表达,NTHi能使EGFR或p38MAPK磷酸化.p38MAPK、EGFR特异性抑制剂或共转染EGFR显性失活质粒,可显著抑制NTHi所诱导的MUC5AC的表达,EGFR特异性抑制剂对NTHi所诱导p38MAPK磷酸化有抑制作用.结论 不分型流感嗜血杆菌可通过EGFR-p38MAPK信号通路,上调人类MUC5AC黏液素基因表达.

abstractsObjective To investigate the signaling mechanisms underlying up-regulation of nontypeable Haemophilus influenzae(NTHi)-induced MUC5AC mucin expression. Methods The expression of MUC 5AC at mRNA level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR)and real-time fluorescent quantitative PCR.Immunoprecipitation and Western blot were performed tO examine the phosphorylation of p38 mitogen-activated protein kinase(p38MAPK)and epidermal growth factor receptor(EGFR)or the effect of dominant negative mutant of EGFR on the phosphorylation of p38MAPK in HM3 cells treated with NTHi.Luciferase assay was also performed to examine the effect of p38MAPK and EGFR inhibitors or dominant negative mutant of EGFR on NTHi-induced MUC5AC expression at transcription level.Results NTHi induced MUC5AC mucin expression at both mRNA and transcription levels.Phosphorylation of p38MAPK and EGFR were observed in HM3 cells treated with NTHi.Either SB203580,a specific inhibitor for p38MAPK or AGl478,a specific inhibitor for EGFR.inhibited NTHi-induced MUC5AC up-regulation at transcription level. Furthermore,Overexpressing dominant negative mutant of EGFR also inhibited NTHi-induced MUC5AC upregulation at transcription 1evel in a dos-dependent manner.EGFR inhibitor reduced NTHi-induced p38MAPK phosphorylation in HM3 cells.Conclusion Bacterium NTHi up-regulates MUC5AC mucin transcription via EGFR-p38MAPK signaling pathway.