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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 main signal nodes of rhythmic pressure wave that maintain the basic secretion of airway mucin (MUC). Methods Human airway epithelial 16HBE cells were cultured by air / liquid interface method. The rhythmic pressure wave generated by normal respiration was cultured in cultured cells and divided into static control group, rhythmic pressure wave group, rhythmic pressure wave + tension sensitivity (TRPV4) antagonist ruthenium red, rhythmic pressure wave + verapamil, rhythmic pressure wave + protein kinase C inhibitor (GF109203x). The changes of Ca2 + concentration were observed by confocal laser scanning microscope TRPV4mRNA expression, West-ern bolt detection of MARCKS and SNAP-23 levels, ELISA detection of MUC (2,5AC and 5B) secretion. Results The intracellular Ca2 + concentration (312.34 ± 17.08) and TRPV4 mRNA (0.63 ± 0.03) in rhythmic stress wave group were significantly higher than those in the static control group (196.16 ± 35.06,0.28 ± 0.05) (P <0.01) P <0.01). At the same time, MUC (2, 5AC and 5B) secreted pMARCKS and pSNAP-23 protein levels significantly increased (P <0.01) .After Ca2 + and PKC inhibitor pretreatment, 0.01). CONCLUSION TRPV4 participates in rhythmic pressure wave to maintain basal MUC secretion balance of airway epithelium. This process can activate Ca2 + / PKC / MARCKS signaling pathway.