ATP过量释放在机械牵张致气道黏液高分泌中的作用

发布时间：2018-11-08 06:58

【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导致的气道黏液高分泌中所起到的作用。方法： (1)人气道上皮细胞(HBE16)行体外培养，在培养板倾斜30°（即1/3细胞无培养液覆盖并暴露于空气中）0、10、15、30、45s时分别行四甲基偶氮唑盐光吸收法(MTT法)测定细胞活性，并确定最佳倾斜时间；（2）通过有节律的倾斜细胞培养板，利用液体表面张力、大气压及液体重力所产生的牵张力及压力(可分解为剪应力与压应力)给予牵张刺激,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；（3）各组细胞依施加条件不同而分为8组：①对照组、②单纯倾斜组、③倾斜+胞内钙离子（Ca~(2+)）螯合剂（BAPTA-AM）、④倾斜+BAPTA-AM+胞外Ca~(2+)螯合剂（EGTA）、⑤加压+倾斜、⑥加压+倾斜+RB-2、⑦加压+倾斜+BAPTA-AM、⑧加压+倾斜+BAPTA-AM+EGTA；（4）分别采用MTT法测定细胞活性、逆转录聚合酶链反应(RT-PCR)检测各组黏蛋白(MUC)5ACmRNA表达水平、酶联免疫反应(ELISA)法检测细胞培养上清液中MUC5AC含量、高效液相色谱(HPLC)法检测培养液中ATP释放量、倒置荧光显微镜观察细胞内Ca~(2+)浓度变化。结果：（1）加压+倾斜组细胞MUC5ACmRNA相对含量、培养上清液中ATP释放量和MUC5AC蛋白分泌量分别为：（11.8±0.01）、（7.41±0.45）μmol/g、(0.77±0.26)，，显著高于单纯倾斜组的(6.6±0.01)、(2.76±0.47)μmol/g、(0.25±0.01)及对照组的(3.4±0.01)、(0.00±0.01）μmol/g、（0.02±0.01）（均P0.05）。（2）RB-2、EGTA+BAPTA-AM处理后的加压+倾斜组细胞的MUC5ACmRNA相对含量分别为：（10.5±0.03)、（11.9±0.11），与加压+倾斜组(11.80±0.01)相比抑制作用不显著（P0.05），但显著高于对照组的（3.40±0.01）（P0.05）。（3）RB-2、EGTA+BAPTA-AM处理后的加压+倾斜组细胞培养上清液中MUC5AC蛋白含量分别为(0.07±0.05),(0.08±0.05),较加压+倾斜组(0.74±0.27)显著降低（均P0.05）。同时，与对照组(0.02±0.01)相比，加压+倾斜组能显著增加人气道上皮细胞培养上清液中MUC5AC蛋白的含量(P0.05)；（4）RB-2、EGTA+BAPTA-AM处理后的加压+倾斜组细胞培养上清液中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[(7.41±0.45)μmol/g]显著降低（均P0.05）。同时，与对照组[(0.00±0.01)μmol/g]相比，加压+倾斜能明显提升细胞培养上清中ATP的含量(P0.05)；（5）培养板倾斜角度最大时，胞内Ca~(2+)浓度达高峰，倾斜组胞内Ca~(2+)浓度峰明显高于对照组，加压+倾斜组内Ca~(2+)浓度峰明显高于倾斜组；给予BAPTA-AM及RB-2预处理后胞内Ca~(2+)浓度峰明显降低，而EGTA预处理后Ca~(2+)浓度峰无明显变化。结论：（1）机械通气可以显著提高气道黏膜上皮MUC5AC的分泌与表达；（2）气道上皮细胞依赖Ca~(2+)的ATP过量释放与气道黏膜上皮MUC5AC的高分泌密切相关，但与MUC5ACmRNA表达的关系不密切；（3）该机制中的Ca~(2+)几乎全部由胞内存储钙提供。
[Abstract]:Aim: to investigate the role of adenosine triphosphate (ATP) release in airway mucus hypersecretion induced by mechanical ventilation. Methods: (1) Human duct epithelial cells (HBE16) were cultured in vitro. When the culture plate tilted 30 掳(that is, 1 / 3 cells were not covered by culture medium and exposed to air), the cell activity was determined by tetramethylazolium photoabsorption assay (MTT) for 45 s, and the optimum tilting time was determined. (2) through a rhythmically inclined cell culture plate, tension is stimulated by the tension and pressure generated by liquid surface tension, atmospheric pressure and liquid gravity (which can be decomposed into shear stress and compressive stress). In order to simulate mechanical ventilation, medical oxygen is injected into the closed box to increase pressure. (3) the cells in each group were divided into 8 groups according to different applied conditions: 1 control group, 2 simple tilt group, 3 tilted intracellular calcium ion (Ca~ (2) chelating agent (BAPTA-AM), 4 tilted BAPTA-AM extracellular Ca~ (2) chelator (EGTA), 5 pressurized tilt, 6 pressurized tilt RB-2,7 pressurized tilt BAPTA-AM,8 pressurized tilt BAPTA-AM EGTA; (4) the cell activity was measured by MTT assay, the expression of mucin (MUC) 5ACmRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR), and the content of MUC5AC in the supernatant of cell culture was detected by enzyme-linked immunosorbent assay (ELISA). High performance liquid chromatography (HPLC) (HPLC) method was used to detect the amount of ATP released in the culture medium, and the concentration of Ca~ (2) was observed by inverted fluorescence microscope. Results: (1) the relative content of MUC5ACmRNA, the release of ATP and the secretion of MUC5AC protein in the culture supernatant were (11.8 卤0.01), (7.41 卤0.45) 渭 mol/g, (0.77 卤0.26), respectively. It was significantly higher than that in tilting group (6.6 卤0.01), (2.76 卤0.47) 渭 mol/g, (0.25 卤0.01) and control group (3.4 卤0.01), (0.00 卤0.01) 渭 mol/g, (0.02 卤0.01) (P0.05). (2) the relative content of MUC5ACmRNA was (10.5 卤0.03), (11.9 卤0.11) in the pressure-inclined group treated with RB-2,EGTA BAPTA-AM. Compared with the pressure tilt group (11.80 卤0.01), the inhibitory effect was not significant (P0.05), but significantly higher than the control group (3.40 卤0.01) (P0.05). (3) the content of MUC5AC protein in the supernatant of the cell culture supernatant treated with RB-2,EGTA BAPTA-AM was (0.07 卤0.05), (0.08 卤0.05). Compared with the pressure tilt group (0.74 卤0.27), significantly decreased (P0.05). At the same time, compared with the control group (0.02 卤0.01), the pressure tilt group significantly increased the content of MUC5AC protein in the supernatant of human duct epithelial cells (P0.05). (4) the amount of ATP released from the supernatant of the cell culture supernatant treated with RB-2,EGTA BAPTA-AM was (0.05 卤0.02) 渭 mol/g, (0.08 卤0.02) 渭 mol/g, respectively. Compared with the pressure tilt group [(7.41 卤0.45) 渭 mol/g], it was significantly lower (P0.05). At the same time, compared with the control group [(0.00 卤0. 01) 渭 mol/g], the ATP content in the supernatant was significantly increased (P0.05). (5) when the tilting angle of culture plate was the largest, the concentration of intracellular Ca~ (2) reached the peak, the concentration of intracellular Ca~ (2) in tilted group was significantly higher than that in control group, and the concentration peak of Ca~ (2) in tilted group was higher than that in tilted group. After pretreatment with BAPTA-AM and RB-2, the concentration peak of Ca~ (2) decreased significantly, but the concentration peak of Ca~ (2) did not change after EGTA pretreatment. Conclusion: (1) Mechanical ventilation can significantly increase the secretion and expression of MUC5AC in airway mucosal epithelium; (2) the excessive release of ATP dependent on Ca~ (2) in airway epithelial cells was closely related to the high secretion of MUC5AC in airway mucosal epithelium, but not to the expression of MUC5ACmRNA. (3) almost all of the Ca~ (2) in this mechanism was provided by intracellular storage calcium.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562

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