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p38MAPK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,井研究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及转录水平上凋人类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EGFRp38MAPK信号通路,上调人类MUC5AC黏液素基因表达。
Objective To investigate the signaling mechanisms underlying up-regulation of nontypeable Haemophilus influenzae(NTHi) induced MUCSAC mucin expression. Methods The expression of MUC5AC 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 MUCSAC 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 AG1478, a specific inhibitor for EGFR, inhibited NTHi-induced MUCSAC up regulation at transcription level. Furthermore, overexpressing dominant negative mutant of EGFR also inhibited NTHi-indueed MUC5AC upregulation at transcription level in a dose-dependent manner. EGFR inhibitor reduced NTHi-induced p38MAPK phosphorylation in HM3 cells. Conclusion Bacterium NTHi up-regulates MUCSAC mucin transcription via EGFR-p38MAPK signaling pathway.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2008年第6期324-328,共5页
Chinese Journal of Infectious Diseases
基金
国家973计划资助项目(2005CB523102)
上海市自然科学基金资助项目(07ZR15014)
美国国立卫生研究院(National Institutes of Health,NIH)基金资助项目(DC004562、DC005843)