中文摘要：

目的：以人中性粒细胞弹性蛋白酶（HNE）为诱导因素，研究建立黏蛋白（MUC）5AC和5B高表达的细胞模型，同时对黏蛋白高表达机制进行初步研究。方法：培养人肺腺癌细胞A549，以HNE为刺激因素，EGFR中和抗体、表皮细胞生长因子受体（EGFR）磷酸化阻断剂AG1478为干预因素，分组培养。采用四甲基偶氮唑盐光吸收法（MTT法）检测HNE对细胞活性的影响；逆转录-聚合酶链反应（RT—PCR）检测MUC5ACmRNA，MUC5BmRNA的变化；酶联免疫吸附测定法（ELISA）定量分析MUC5AC和MUC5B蛋白含量的差异；细胞免疫化学以及激光共聚焦技术进一步直观观察MUC5AC，MUC5B，p-EGFR蛋白表达的变化。结果：HNE对A549细胞活力的影响呈剂量依赖性；HNE刺激组的MUC5AC，MUC5B基因转录和蛋白表达水平均明显高于对照组，差异有统计学意义（均P〈0.01）；HNE刺激组P-EGFR蛋白表达显著增多，EGFR中和抗体、AG1478能显著降低HNE诱导的MUC5AC高表达，但对MUC5B高表达无干预作用。结论：人肺腺癌细胞A549同时表达MUC5AC和MUC5B，HNE能有效刺激A549细胞高表达MUC5AC和MUC5B，黏蛋白高表达细胞模型的建立为研究气道粘液高分泌疾病提供了实验基础。HNE通过激活EGFR信号转导通路诱导MUC5AC的高表达，但MUC5B高表达机制与之不同，有待进一步研究。

英文摘要：

Objective： To investigate the establishment of the experimental cell model of MUCSAC and MUCSB high expression induced by human neutrophil elastase （HNE） and the relative mechanism. Methods： Human lung adenocarcinoma A549 cells were cultured and divided into six groups：control group, HNE25nmol/L group, HNE50nmol/L group, HNE100nmol/L group, anti-EGFR antibody ＋ HNE50nmol/L group and AG1478 ＋ HNE50nmol/L group. The cell activity was assessed by MTT method. The changes of MUCSAC mRNA and MUCSB mRNA were analyzed by RT-PCR. The protein expression changes of MUCSAC and MUCSB were detected by ELISA, while the protein expression changes of MUCSAC, MUCSB and p-EGFR were observed by Immunofluorescence and confocal laser technology. Results： HNE depressed the cell activity a dose-dependent manner. The gene transcription and the protein expression of MUCSAC and MUCSB in the HNE-induced groups were significantly higher than those in the control group and the differences were statistically significant （P 〈 0.01 ）. p-EGFR protein expression in the HNE50nmol/L group was much more than that in the control group. MUCSAC mRNA and protein in the anti- EGFR antibody ＋ HNE50nmol/L group and the AG1478 ＋ HNE50nmol/L group were lower than those in the HNE50nmol/L group, but MUCSB mRNA and protein had no differences between these groups. Conclusion： Human lung adenocarcinoma A549 cells express MUCSAC and MUCSB simultaneously, and HNE can effectively stimulate the high expression of MUCSAC and MUCSB. The A549 cell with mucin high expression induced by HNE can provide the cell model, which is similar with the environment in vivo and helps the research of airway mucus hypersecretion disease. HNE induces the high expression of MUCSAC via EGFR signal transduction pathway, but MUCSB high expression mechanism is different, which need further study.