CAJ | 학술논문

目的观察张力敏感性阳离子通道、Ca2+内流和豆蔻酰化富丙氨酸C激酶底物(MARCKS)在机械牵张引起气道黏液高分泌中的作用.方法人气道黏膜上皮细胞(HBE16)体外培养,采用小型生物撞击机给予机械牵张刺激,各组培养细胞依施加条件不同而分为对照、牵张、牵张+钆、牵张+硝苯吡啶、牵张+低分子量肝素、牵张+MARCKS效应结构域(ED)锁核酸(LNA)以及牵张+MARCKS的ED无关对照LNA序列共7组,分别采用逆转录聚合酶链反应(RT-PCR)和免疫荧光法观察与胞吐相关的突触相关膜蛋白SNAP23以及黏蛋白(MUC)5AC mRNA和蛋白表达,酶联免疫吸附试验(ELISA)方法检测细胞培养上清中MUC5AC分泌.结果机械牵张能显著升高人气道黏膜上皮细胞中SNAP23和MUC5AC表达,显著提升细胞培养上清中MUC5AC分泌(P＜0.05);钆、硝苯吡啶、MARCKS的ED-LNA均能抑制机械牵张对SNAP23表达和MUC5AC表达、分泌的提升作用(均P＜0.05);而低分子量肝素未能显著降低SNAP23表达和MUC5AC表达、分泌(P＞0.05).结论机械牵张能升高人气道黏膜上皮细胞MUC5AC的表达,其机制可能与张力敏感性阳离子通道、Ca2+内流和MARCKS途径有关.
목적 관찰장력민감성양리자통도、Ca2+내류화두구선화부병안산C격매저물(MARCKS)재궤계견장인기기도점액고분비중적작용.방법 인기도점막상피세포(HBE16)체외배양,채용소형생물당격궤급여궤계견장자격,각조배양세포의시가조건불동이분위대조、견장、견장+구、견장+초분필정、견장+저분자량간소、견장+MARCKS효응결구역(ED)쇄핵산(LNA)이급견장+MARCKS적ED무관대조LNA서렬공7조,분별채용역전록취합매련반응(RT-PCR)화면역형광법관찰여포토상관적돌촉상관막단백SNAP23이급점단백(MUC)5AC mRNA화단백표체,매련면역흡부시험(ELISA)방법검측세포배양상청중MUC5AC분비.결과 궤계견장능현저승고인기도점막상피세포중SNAP23화MUC5AC표체,현저제승세포배양상청중MUC5AC분비(P＜0.05);구、초분필정、MARCKS적ED-LNA균능억제궤계견장대SNAP23표체화MUC5AC표체、분비적제승작용(균P＜0.05);이저분자량간소미능현저강저SNAP23표체화MUC5AC표체、분비(P＞0.05).결론 궤계견장능승고인기도점막상피세포MUC5AC적표체,기궤제가능여장력민감성양리자통도、Ca2+내류화MARCKS도경유관.
Objective The effects of stretch sensitive positive ion channel, the internal flow of Ca2+ and myristoylated alanine-rich C kinase substrate (MARCKS) on the high externalization of MUC5AC in airway epithelial cells in mechanical stretching. Methods Mini-type Multi-fuctional Bio-impact Machin was used to construct the model of mechanical stretching. The airway epithelial cells were divide into control group, stretch, stretch and gadolinium , stretch and low molecular weight heparin, stretch and nifedipine ,stretch and the locked nucleic acids of MARCKS effective domain ( ED), stretch and the control locked nucleic acids of MARCKS ED groups. The expression of MUC5AC and SNAP23 protein in cells were determined by immunohistochemistry method, contents of MUC5AC and SNAP23 protein were measured by Image Pro Plus 5.0. MUC5AC protein in supernatant was determined by ELISA methods. SNAP23 mRNA was determined by RT-PCR methods. Results Mechanical stretching could increase the expression of SNAP23 and MUC5AC in cells and MUC5AC in supernatant( P ＜ 0. 05). Gadolinium , nifedipine and LNA of MARCKS ED could reduce the increase the expression of SNAP23 and MUC5AC in cells and MUCSAC in supernatant(all P ＜0. 05). ConclusioN Mechanical stretching could increase the expression of MUC5AC in airway epithelial cells. This maybe is concerned with stretch sensitive positive ion channel, the internal flow of Ca2+ and MARCKS.