吉非替尼对慢性阻塞性肺疾病黏液高分泌的影响

Effect of gefitinib on mucus hypersecretion in chronic obstructive pulmonary disease

目的探讨应用吉非替尼抑制表皮生长因子受体（EGFR）酪氨酸激酶活性对慢性阻塞性肺疾病（COPD）黏液高分泌的影响。方法采用香烟提取物（CSE）刺激支气管上皮细胞株16HBE制备COPD模型，采用EGFR酪氨酸激酶抑制剂吉非替尼抑制模型细胞的EGFR自身酪氨酸位点磷酸化，实时定量聚合酶链反应（realtimePCR）检测EGFR、黏蛋白MUC5AC基因水平，Westernblot检测EGFR、磷酸化EGFR（PEGFR）蛋白表达水平，酶联免疫吸附法（ELISA）检测MUCSAC蛋白水平。结果与对照组比较，CSE组、吉非替尼组的EGFR基因表达分别上调12．7％、8．6％，差异均无统计学意义（P〉0．05）；MUCSAC基因水平分别上调141．7％、26．4％，差异有统计学意义（P〈0．05）。对照组、CSE组、吉非替尼组EGFR蛋白相对含量分别为600．34±64．58、632．58±72．94、584．57±67．39，各组之间比较差异无统计学意义（P〉0．05）。p-EGFR蛋白相对含量分别为338．62±45．28、679．43±78．23、292．74±59．17，CSE组p-EGFR蛋白含量较对照组显著增加（P〈0．05），吉非替尼组PEGFR蛋白水平与对照组比较差异无统计学意义（P〉0．05）。对照组、CSE组、吉非替尼组MUC5AC蛋白含量分别为（72．80±6．25）μg／mg、（187．00±10．26）μg／mg，（92．57±8．32）μg／mg，与对照组比较，CSE组MUCSAC蛋白表达显著增加（P〈0．05），吉非替尼组MUC5AC蛋白水平与对照组比较差异无统计学意义（P〉0．05）。结论香烟烟雾可能通过EGFR途径导致气道黏液过度分泌，应用EGFR受体酪氨酸激酶抑制剂吉非替尼可通过抑制EGFR自身酪氨酸激酶活性，有效阻断香烟烟雾诱发的气道黏液高分泌，EGFR作为COPD治疗的一个靶点可行有效。

Objective To investigate the inhibiting epidermal growth factor receptor （EGFR） effect of gefitinib on mucus hypersecretion by activity in chronic obstructive pulmonary disease （COPD）. Methods Human airway epithelail cell lines 16HBE cells were exposed to cigarette smoke extraction （CSE） to establish the COPD model. EGFR activity was inhibited by tyrosine kinase inhibitor gefitinib. The mRNA expressions of EGFR and MUC5AC were detected by real-time PCR. EGFR, p-EGFR and MUCSAC protein levels were determined by Western blot and ELISA. Results EGFR mRNA level was increased by 12.7% in CSE and 8.6% in gefitinib group, but had no significant differences among CSE, gefitinib group and control group （all P〉 0.05）. MUC5AC mRNA levels were enhanced by 141.7%,26.4% in CSE group and gefitinib group respectively, and there were significant differences among CSE, gefitinib group and control group （all P〈0.05）. EGFR protein levels were （600.34±64.58） μg/mg, （632.58±72.94）μg/mg, （584.57±67.39）μg/mg, in control, CSE and gefitinib groups, respectively, and there were no significant differences between groups （all P〉0.05）. p-EGFR protein levels were （338.62±45.28）μg/mg, （679.43±78.23）μg/mg, （292.74±59. 17）μg/mg in control, CSE and gefitinib groups, respectively. MUC5AC protein levels were（72.80±6.25）μ/mg, （187.00±10.26）μg/mg, （92.57＋8.32）μg/mg in control, CSE and gefitinib groups respectively. Compared with control group, p-EGFR and MUC5AC protein levels were increased significantly in CSE group （both P〈0. 05）, and had no significant differences in p EGFR and MUC5AC protein levels between control group and gefitinib group. Conclusions CSE may lead to mucus hypersecretion through activating the EGFR-mediated signaling pathways. Gefitinib may inhibit mucus hypersecretion by inhibiting EGFR tyrosine kinanse activity. EGFR may serve as a potential target for COPD.