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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。
In order to observe the effect of Staphylococcus aureus enterotoxin B (SEB) on the secretion of mucin MUC5AC induced by nasal drilling rhombial epithelial cells and to explore its mechanism, we first cultured human nasal mucosal epithelial cell line HNEpC in vitro (SEB). The expression of MUC5AC and TGF-α in culture supernatants were detected by ELISA. The expression of MUC5AC mRNA was detected by real-time quantitative PCR. The expression of epidermal growth factor receptor (EGFR ) Phosphorylation. At the same time, TALE siRNA was used to interfere the expression of BIS and its effect on TGF-α secretion. Finally, the cells were treated with TGF-α neutralizing antibody, EGFR inhibitor AG-1478 or TALE inhibitor TAPI, Mediate the role of MUC5AC secretion. The results showed that SEB secreted MUC5AC and expressed its mRNA after 1, 10, and 100 ng / mL SEB treatment for 24 h, and its secretion level increased with the increase of SEB concentration or time The extension of the gradual increase. SEB can also induce nasal epithelial cells to secrete TGF-α and phosphorylate EGFR. After TAPI treatment with 10μmol / L TALE inhibitor, the levels of TGF-α and MUC5AC were significantly decreased, and similar results were obtained for TGF-α and MUC5AC after interference with TALE siRNA. Pre-incubation of cells with TGF-α neutralizing antibody for 30 min significantly inhibited EGFR phosphorylation. In addition, TGF-α and EGFR neutralizing antibodies as well as AG-1478 pretreatment also decreased MUC5AC secretion. The above results showed that SEB induced MUC5AC expression in human nasal epithelial cells via TALE / TGF-α / EGFR.