依赖Ca2+的ATP过量释放在机械牵张所致气道上皮黏蛋白5AC高分泌中的作用

摘要

目的 探讨人气道上皮细胞三磷酸腺苷(ATP)释放量的改变在机械通气所致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,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)给予压力牵张刺激,并利用向密闭盒中注入医用氧气增加压力以模拟机械通气,各组培养细胞依施加条件不同而分为对照组、单纯倾斜组、倾斜+钙离子螯合剂BAPTA-AM、倾斜+BAPTA-AM+钙离子螯合剂EGTA、加压+倾斜、加压+倾斜+嘌呤P2Y受体阻断剂reactive blue-2(RB-2)、加压+倾斜+ BAPTA-AM、加压+倾斜+ BAPTA-AM +EGTA,共8组,分别采用四甲基偶氮唑盐法(MTT)测定细胞活性、逆转录聚合酶链反应(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含量、高效液相色谱法(HPLC)检测培养液中ATP释放量.结果 加压+倾斜组细胞MUC5AC mRNA相对含量、培养上清液中ATP释放量和MUC5AC蛋白分泌量分别为:(11.80±0.01)、(7.41±0.45)μmol/g、(0.77±0.26),显著高于单纯倾斜组的(6.60±0.01)、(2.76±0.47)μmol/g、(0.25±0.01)及对照组的(3.40±0.01)、(0.00±0.01)μmol/g、(0.02±0.01)(均P＜0.05).RB-2、EGTA + BAPTA-AM处理后的加压+倾斜组细胞的MUC5AC mRNA相对含量分别为:(10.5±0.03)、(11.9±0.11),与加压+倾斜组相比抑制作用不显著(P＞0.05);培养上清液中ATP释放量分别为:(0.05±0.02)μmol/g、(0.08±0.02)μmol/g,较加压+倾斜组显著降低(均P＜0.05).结论机械通气可以显著提高气道黏膜上皮MUC5AC的分泌,其机制与气道上皮细胞依赖Ca2+的ATP的过量释放密切相关.%Objective To explore the effects of Ca2+-dependent ATP over-release on mucin(MUC) 5 AC over-externaliza-tion in airway epithelial cells by mechanical stretching. Methods Airway epithelial cells were stimulated by rhythmically tilting the culture dish. The shear stress and compressive stress were provided by liquid surface tension,atmospheric pressure and liquid gravity. Medical oxygen was injected into closed boxes to increase tension to simulate mechanical ventilation. The airway epithelial cells(HBE16) were divided into control group, tilt group, tilt+ Ca2+-chelator BAPTA-AM group, tilt+BAPTA-AM + Ca2+-chelator EGTA group,tilt+air pressure group,tilt+air pressure+P2Y receptor blocking pharmacon [reactive blue-2(RB-2)] group, tilt+pressure+BAPTA-AM group, tilt+air pressure+BAPTA-AM+EGTA group. The levels of MUC5AC protein and ATP in culture medium,and MUC5AC mRNA in culture cells were detected by using enzyme-linked immunosorbent assay,high performance liquid chromatography and RT-PCR,respectively. Results The expression levels of MUC5AC mRNA in culture cells,its protein and ATP in culture medium of tilt +air pressure group were( 11. 80 + 0. 01),(7. 41 + 0. 45)μmol/g and(0. 77 + 0. 26)respectively,which were increased significantly(all P<0. 05)as compared with those in tilt group [(6. 60 + 0. 01), (2. 76 + 0. 4 7),μmol/g,(0. 25 + 0. 01)] and control group [(3. 40 + 0. 01) , (0. 00 + 0. 01)μmol/g, (0. 02 + 0. 01)]. After the cells were pre-treated with RB-2 ,EGTA + BAPTA-AM in tilt + air pressure group, the mRNA expression [(10. 5 + 0. 03) and(ll. 9 + 0. 11)] was not significantly decreased as compared with tilt+air pressure groupCall P>0. 05) ,but the concentration of ATP in the supernatant [(0. 05 + 0. 02)μmol/g, (0. 08 + 0. 02)μmol/g] was significantly decreased as compared with tilt+ air pressure group(all P<0. 05). Conclusion Mechanical ventilation could increase the secretion of MUC5AC in airway epithelial cells, which may be closely related with Ca2+ -dependent ATP over-release.