金黄色葡萄球菌肠毒素B诱导人鼻黏膜上皮细胞表达黏蛋白MUC5AC的分子机制  

Molecular Mechanism of Staphylococcus aureus Enterotoxin B Induced Secretion of MUC5AC in Human Nasal Epithelial Cells

作　　者：刘珺[1] 廖剑绚[1] 唐洪波[1] 周芝芳[1] 

机构地区：[1]南华大学附属第一医院耳鼻喉科,衡阳421001

出　　处：《基因组学与应用生物学》2016年第6期1277-1283,共7页Genomics and Applied Biology

基　　金：湖南省科技计划项目(2013SK3114);湖南省分子靶标新药研究协同创新中心资助项目(湘教通2015351)共同资助

摘　　要：为了观察金黄色葡萄球菌肠毒素B（SEB）对诱导人鼻钻膜上皮细胞分泌粘蛋白MUC5AC的影响,并初步探讨其作用机制,本研究首先在体外培养人鼻黏膜上皮细胞株HNEpC,用不同浓度的SEB孵育细胞0~24 h,ELISA检测培养上清中MUC5AC和转化生长因子-α（TGF-α）的含量;实时定量PCR检测MUC5AC mRNA表达水平;Western blot分析表皮生长因子受体（EGFR）的磷酸化。同时采用肿瘤坏死因子转化酶（TALE）siRNA干扰其表达双察其对TGF-α分泌的影响最后采用TGF-α中和抗体、EGFR抑制剂AG-1478或TALE抑制剂TAPI处理细胞,观察其在介导MUC5AC分泌中的作用。结果显示,1 ng/mL、10 ng/mL和100 ng/mL SEB作用鼻黏膜上皮细胞24 h后,可明显诱导其分泌MUC5AC并表达其mRNA,且其分泌水平随着SEB浓度的增高或时间的延长而逐渐增多。SEB也能诱导鼻黏膜上皮细胞分泌TGF-α,并磷酸化EGFR。给予10μmol/L TALE抑制剂TAPI处理细胞后,TGF-α和MUC5AC分泌水平显著减少,采用TALE siRNA干扰其表达后,TGF-α和MUC5AC也得到了类似结果。采用TGF-α中和抗体预孵育细胞30 min后,可明显抑制EGFR磷酸化。此外,TGF-α和EGFR中和抗体以及AG-1478预处理也可降低MUC5AC分泌。以上结果表明SEB经TALE/TGF-α/EGFR诱导人鼻黏膜上皮细胞表达MUC5AC。The aim of this study is to investigate the effect of the Staphylococcus aureus Enterotoxin B（SEB）stimulated secretion of MUC5AC in human nasal epithelial cells and discuss its molecular mechanism.In the first place,we cultured the human nasal epithelial cell line HNEpC in vitro,and then cells were stimulated with different concentrations of SEB for 0~24 h.The contents of MUC5AC and TGF- a in culture supernatant were detected by ELISA.Real-time quantitative PCR was used to detect the mRNA expression of MUC5AC.Phosphorylation of epithelial growth factor receptor（EGFR） was detected by Western blot.The secretion of TGF-α was detected when tumor necrosis factor converting enzyme（TACE） was knockdown by siRNA.Cells were pre-treated by antibody of TGF- a,inhibitor of EGFR（AG-1478） or inhibitor of TACE（TAPI） to observe the involvement of TGF-α,EGFR or TACE in secretion of MUC5A.Results indicated that human nasal epithelial cells pretreated with 1 ng/mL,10 ng/mL and 100 ng/mL SEB for 24 h could induce the secretion of MUC5 AC and expression of mRNA,as the increase of the concentration of SEB or time interval,the level of MUC5AC concentration and mRNA were increased.Same results were obtained from the real-time PCR.SEB could stimulate the secretion of TGF-α and phosphorylation of EGFR in nasal epithelial cells.The expression of TGF-a and MUC5AC were significantly decreased when the cells were pretreated with TAPI or the TACE siRNA.Pretreatment with the neutralizing antibody against EGFR for 30 min could prevent the phosphorylation of EGFR.Furthermore,pretreatment with the neutralizing antibody against EGFR and TGF-α or AG-1278 could decrease secretion of MUC5AC.These results indicate that SEB induces MUC5AC secretion via TACE/TGF-α/EGFR pathway in human nasal epithelial cells.

关 键 词：金黄色葡萄球菌肠毒素B 表皮生长因子受体 肿瘤坏死因子α转化酶 转化生长因子-Α 粘蛋白5AC 

分 类 号：R765[医药卫生—耳鼻咽喉科]