张力敏感性阳离子通道在气道黏液基础分泌中的介导作用

The mediated effect of tension-sensitive cation channel in airway basic mucus secretion

目的探讨节律性压力波维持气道黏蛋白(MUC)基础性分泌的主要信号节点。方法采用气/液相界面法培养人气道上皮16HBE细胞,模拟正常呼吸所产生的节律性压力波作用于培养细胞,分为静态对照组、节律性压力波组、节律性压力波+张力敏感性阳离子通道(TRPV4)拮抗剂钌红、节律性压力波+维拉帕米、节律性压力波+蛋白激酶C抑制剂(GF109203x)共5组,通过激光共聚焦观察Ca2+浓度变化,RT-PCR检测TRPV4mRNA表达,West-ern bolt检测MARCKS及SNAP-23水平,ELISA检测MUC(2、5AC和5B)分泌情况。结果节律性压力波组胞内Ca2+浓度(312.34±17.08)和TRPV4 mRNA(0.63±0.03)较静态对照组(196.16±35.06,0.28±0.05)显著增高(P<0.01),RR干预后明显降低(P<0.01);同时MUC(2、5AC和5B)分泌,pMARCKS及pSNAP-23蛋白水平明显增高(P<0.01),给予Ca2+及PKC抑制剂预处理后,上述各指标蛋白均降低(P<0.01)。结论 TRPV4参与节律性压力波维持气道上皮基础性MUC分泌平衡,此过程籍以激活Ca2+/PKC/MARCKS信号通路实现。

Objective To investigate the possible signal transduction pathways of rhythmic pressure maintained airway mucous secretion in airway epithelial cells.Methods Using gas/liquid interface to culture human airway epithelial 16HBE cells in vitro,two devices mimicked normal pressure wave which generated by the respiratory rhythm and affected the cultured cells.16HBE cells were divided into control group,rhythmic pressure,pressure and Ruthenium red,pressure and Verapamil,pressure and PKC inhibitors（GF109203x）.The Ca2＋concentration was observed by laser confocal,the level of TRPV_4 mRNA,the relative contents of pMARCKS and pSNAP-23,and the secretion of MUC（2,5AC and 5B） in culture medium were detected by RT-PCR,Western blot and ELISA respec-tively.Results Intracellular Ca2＋ concentration（312.34±17.08） and TRPV_4 mRNA（0.63±0.03） level in the rhythmic pressure group were significantly higher than in the control group（P0.01）,which decreased markedly after the intervention of RR（P0.01）,meanwhile,there were obvious increases in the secretion of MUC（2,5AC and 5B）,the relative contents of pMARCKS and pSNAP-23 protein.After the preprocessing of Verapamil and PKC specific inhibitors,either of MUC level,pMARCKS and pSNAP-23 protein reduced（P0.01）.ConclusionsRhythmic pressure maintained the secretion of MUC in airway epithelial by activation of TRPV_4/Ca2＋/PKC/MARCKS signal pathway.