上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (12): 1667-.doi: 10.3969/j.issn.1674-8115.2011.12.002

• 论著（基础研究） • 上一篇    下一篇

气道上皮细胞ATP释放在周期性压力所致气道黏液分泌中的作用

苏晖晖, 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:2011-12-28 发布日期:2012-01-04
  • 通讯作者: 周向东, 电子信箱: zxd999@263.net。
  • 作者简介:苏晖晖（1985—）, 女, 硕士生；电子信箱: suhuih@126.com。
  • 基金资助:

    国家自然科学基金(31171346)；国家自然科学基金中俄国际合作项目（81011120108）

Role of ATP release in cyclic pressure-induced mucin excretion in airway epithelial cells

SU Hui-hui, ZHOU Xiang-dong   

  1. Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400010, China
  • Online:2011-12-28 Published:2012-01-04
  • Supported by:

    National Natural Science Foundation of China, 31171346；Cooperation Project of China and Russia of National Natural Science Foundation of China, 81011120108

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摘要：

目的 探讨气道上皮细胞三磷酸腺苷(ATP)的释放及胞内钙离子(Ca2+)在周期性压力波所致气道黏液分泌中的作用。方法 体外培养人支气管上皮细胞株16HBE，通过节律性倾斜细胞培养板，利用液体的表面张力、大气压及液体重力所产生的牵张力(剪应力)及压力(压应力)给予细胞机械刺激。根据对培养细胞所施加条件不同分为对照组、单纯倾斜组、倾斜+ Ca2+螯合剂BAPTA-AM组、倾斜+ Ca2+螯合剂EGTA组和倾斜+P2Y受体阻断剂RB-2组。分别采用四甲基偶氮唑盐(MTT)法测定各组细胞存活率，反转录聚合酶链反应(RT-PCR)检测各组黏蛋白(MUC)5AC mRNA表达水平，ELISA法检测细胞培养上清液中MUC5AC蛋白含量，高效液相色谱法(HPLC)检测培养液中ATP释放量，倒置荧光显微镜观察细胞内Ca2+浓度变化。结果 与对照组相比，单纯倾斜组16HBE细胞MUC5AC mRNA和蛋白的表达量及细胞培养上清液中ATP的含量均明显升高(P<0.05)；倾斜+BAPTA-AM组、倾斜+RB-2组 ATP及MUC5AC蛋白表达量均明显低于单纯倾斜组（P＜0.05）；倾斜+EGTA 组和倾斜+RB-2组MUC5AC mRNA表达量与单纯倾斜组比较，差异无统计学意义（P>0.05）。结论 节律性压力可刺激气道黏膜上皮MUC5AC分泌，其机制与依赖Ca2+的ATP释放密切相关；该机制中的Ca2+几乎全部由胞内存储钙提供。

关键词: 三磷酸腺苷, 钙离子, 黏蛋白, 周期性压力

Abstract:

Objective To investigate the role of adenosine triphosphate (ATP) release and intra-cellular calcium ion (Ca2+) on cyclic pressure-induced mucin (MUC) excretion in airway epithelial cells. Methods Human bronchial epithelial cell line 16HBE cultured in vitro were stimulated mechanically through tilting the culture dish, and the shear stress and compressive stress were provided by liquid surface tension, atmospheric pressure and liquid gravity. The airway epithelial cells were divided into control group, tilt group, tilt+Ca2+ chelant BAPTA-AM group, tilt+Ca2+ chelant EGTA group and tilt+P2Y receptor blocker RB-2 group. Cell survival rate was measured by MTT assay, expression of MUC5AC mRNA was detected by RT-PCR, expression of MUC5AC protein in culture supernatant was determined by ELISA, ATP release was examined by high performance liquid chromatography (HPLC), and intra-cellular Ca2+ concentration was observed by inverted fluorescence microscopy. Results Compared with control group, the expression of MUC5AC mRNA and protein in 16HBE cells and ATP content in the culture supernatant in tilt group were significantly higher (P<0.05). The expression of MUC5AC protein and ATP content in tilt+BAPTA-AM group and tilt+RB-2 group were significantly lower than those in tilt group (P<0.05). The expression of MUC5AC mRNA in tilt+EGTA group and tilt+RB-2 group was not significantly different from that in tilt group (P>0.05). Conclusion Cyclic mechanical stimulation induces ATP release and MUC5AC excretion in airway epithelial cells in a Ca2+ dependent manner.

Key words: adenosine triphosphate, calcium ion, mucins, cyclic pressure