上海交通大学学报（医学版）

›› 2010, Vol. 30 ›› Issue (1): 59-.

• 论著（基础研究） • 上一篇    下一篇

透明质烷和CD44参与调节气道黏液高分泌的分子机制

余红梅, 周向东   

  1. 重庆医科大学附属第二医院呼吸内科, 重庆 400010
  • 出版日期:2010-01-26 发布日期:2010-01-26
  • 作者简介:余红梅（1982—）, 女, 博士生；电子信箱: yhm-023@sohu.com。
  • 基金资助:

    国家自然科学基金（30770951）

Molecular mechanism of hyaluronan and CD44 on regulation of airway mucous hypersecretion

YU Hong-mei, ZHOU Xiang-dong   

  1. Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
  • Online:2010-01-26 Published:2010-01-26
  • Supported by:

    National Natural Science Foundation of China, 30770951

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摘要：

目的 了解透明质烷（HA）和CD44在气道黏液高分泌中的作用，探索信号因子活化表皮生长因子（EGF）/表皮生长因子受体（EGFR）信号通路的分子机制。方法 体外培养BEAS-2B气道上皮细胞，给予中性粒细胞弹性蛋白酶（NE）刺激，以活性氧（ROS）清除剂（DMTU）、透明质酸酶（Hase）、CD44抗体和组织激肽释放酶抑制剂（PI）作为干预因素。RT-PCR检测黏蛋白（MUC）5AC mRNA表达。ELISA法检测MUC5AC及EGF蛋白含量；Western blotting分析磷酸化EGFR（p-EGFR）蛋白水平。结果 NE刺激后MUC5AC、EGF、pEGFR蛋白及MUC5AC mRNA表达均较对照组显著增高（P<0.01），给予DMTU干预后上述指标水平均明显降低（P<0.01）；在加入DMTU后用Hase处理细胞，上述指标水平均较DMTU组明显升高（P<0.05），EGF蛋白含量增高更为明显（P<0.01）；CD44抗体或PI处理后上述指标水平均明显低于NE组（P<0.05），EGF蛋白含量降低更为显著（P<0.01）。结论 NE可刺激细胞产生ROS，后者可裂解HA致其解聚，并使之与CD44结合而活化组织激肽释放酶（TK），由活化的TK对前体EGF加工处理成EGF，由此启动EGFR信号级联反应，上调MUC5AC基因的表达。

关键词: 透明质烷, CD44, 活性氧, 黏蛋白

Abstract:

Objective To investigate the effects of hyaluronan (HA) and CD44 on airway mucous hypersecretion, and explore the molecular mechanism of activation of epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signal transduction pathway by signal factors. Methods BEAS-2B airway epithelial cells were cultured in vitro, and were stimulated by neutrophil elastase (NE). Reactive oxygen species (ROS) scavenger (DMTU), hyaluronidase (Hase), CD44 antibody and tissue kallikrein (TK) inhibitor (PI) were served as interventional factors, and control group (serum free culture), NE stimulation group (50 nmol/L NE), DMTU+NE group (20 μmol/L DMTU+50 nmol/L NE), DMTU+Hase+NE group (20 μmol/L DMTU+10 μg/mL Hase+50 nmol/L NE), CD44Ab+NE group (5 μg/mL CD44Ab+50 nmol/L NE) and PI+NE group (100 μg/mL PI+50 nmol/L NE) were established. The expression of mucin (MUC) 5AC mRNA was detected by RT-PCR, the expression of MUC5AC and EGF protein was determined by ELISA, and the expression of phosphorylated epidermal growth factor receptor (p-EGFR) protein was analysed by Western blotting. Results The expression of MUC5AC, EGF and p-EGFR protein and MUC5AC mRNA in NE stimulation group was significantly higher than that in control group (P<0.01), the expression in DMTU+NE group was significantly lower than that in NE stimulation group (P<0.01), the expression in MTU+Hase+NE group was significantly higher than that in DMTU+NE group (P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA, and P<0.01 for EGF protein), the expression in CD44Ab+NE group and PI+NE group was significantly higher than that in NE stimulation group (P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA, and P<0.01 for EGF protein). Conclusion NE upregulates the expression of MUC5AC gene via OS/HA/CD44/TK/EGF/EGFR signal transduction pathway in airway epithelial cells.

Key words: hyaluronan, CD44, reactive oxygen species, mucin