机械牵张对气道黏膜上皮细胞黏蛋白表达的影响及其信号通路研究

发布时间：2018-06-06 06:21

本文选题：机械牵张 + 张力敏感性阳离子通道　；参考：《重庆医科大学》2011年博士论文

【摘要】：目的分别观察呼吸机机械通气、机械牵张对兔气道黏膜和人气道黏膜上皮细胞(HBE16)黏蛋白(MUC)5AC表达的影响,并对其信号通路机制进行初步探讨。方法日本大耳兔随机分为对照组、机械通气1、3、6、12和24h组以及2、5和10cmH2O呼气末正压(PEEP)组。取支气管肺泡灌洗液(BALF),用ELISA和RT-PCR法检测BALF中肿瘤坏死因子(TNF)-α、白介素(IL)-8、MUC5AC蛋白和mRNA的水平,计数BALF中多型核粒细胞,底物检测法测定中性粒细胞弹力酶(NE)活性。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,采用流式细胞仪检测牵张前后胞内游离Ca~(2+)荧光强度的变化,激光共聚焦显微镜观察胞内游离Ca~(2+)分布的情况,并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。再将构建的人气道黏膜上皮细胞牵张模型,分别给予丝裂素活化蛋白激酶(MAPK)信号通路的三条主要通路抑制剂:JNK抑制剂(SP600125)、p38蛋白激酶抑制剂(SB203580)及ERK1/2抑制剂(U0126)(浓度均为30μmol/L),并设未进行机械牵张的对照组和单纯机械牵张组。分别采用RT-PCR和ELISA方法检测MUC5AC mRNA表达和蛋白分泌量。结果机械通气能显著增强MUC5AC的分泌(P0.05)。通气3h TNF-α表达至峰值,随后逐渐下降(P0.05)。通气6h IL-8表达至峰值,随后逐渐下降(P0.05)。通气12h后多型核粒细胞数及中性粒细胞弹力酶活性显著升高,24h后回降(P0.05)。随着PEEP的增加,TNF-α、IL-8、MUC5ACmRNA的表达和蛋白含量、多型核粒细胞数和NE活性均随之升高(P0.05)。机械牵张能显著升高人气道黏膜上皮细胞胞内Ca~(2+)浓度(P0.01);同时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(P0.05);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P0.05);而低分子量肝素未能显著降低SNAP23表达和MUC5AC表达、分泌(P0.05)。U0126和SB203580能抑制牵张所致MUC5AC mRNA和蛋白表达增加(均P0.01)。结论机械通气和牵张能促进气道黏膜上皮细胞MUC5AC mRNA的表达和MUC5AC的分泌,其机制可能与机械通气和牵张引发的炎症反应、张力敏感性阳离子通道、Ca~(2+)内流、MARCKS途径以及ERK1/2、p38 MAPK信号通路有关。
[Abstract]:Objective to observe the effects of ventilator mechanical ventilation and mechanical stretch on the expression of mucin MUC5AC in rabbit airway mucosal epithelial cells (HBE16) and to explore its signal pathway mechanism. Methods Japanese large ear rabbits were randomly divided into two groups: control group, mechanical ventilation group of 12 and 24 hours after mechanical ventilation, and group of 2 and 10cmH2O positive end expiratory pressure (PEEP). Bronchoalveolar lavage fluid (BALF) was used to detect the levels of tumor necrosis factor TNF- 伪 (TNF- 伪), interleukin (IL-8) MUC5AC protein and mRNA in BALF by ELISA and RT-PCR methods. The polymorphonuclear cells in BALF were counted and the activity of neutrophil elastase was determined by substrate assay. In vitro culture of human mucosal epithelial cells (HBE16) was performed with a small biological impact machine. The cultured cells in each group were divided into two groups according to different applied conditions: distraction gadolinium and nifedipine. There were 7 groups of low molecular weight heparin, distraction of MARCKS effect domain EDN) and Ed unrelated control LNA sequence of distraction MARCKS. Flow cytometry was used to detect the fluorescence intensity of intracellular free Ca~(2 before and after distraction. Laser confocal microscopy was used to observe the distribution of intracellular free Ca~(2. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the expression of synaptic associated membrane protein SNAP23 and mucin MUC5AC mRNA and protein, respectively. Enzyme linked immunosorbent assay (Elisa) was used to detect the secretion of MUC5AC in the supernatant of cell culture. Then the human airway epithelial cell stretch model was constructed. The mitogen-activated protein kinase (MAPK) signaling pathway was treated with three major pathway inhibitors: 1 / JNK inhibitor SP600125 / p38 protein kinase inhibitor / SB203580) and ERK1/2 inhibitor (30 渭 mol / L) respectively. The control group without mechanical stretch and the control group with mechanical distraction alone were set up. MUC5AC mRNA expression and protein secretion were detected by RT-PCR and ELISA. Results Mechanical ventilation could significantly enhance the secretion of MUC5AC (P 0.05). The expression of TNF- 伪 reached its peak at 3 h after ventilation, and then decreased gradually. The expression of IL-8 reached its peak at 6 h after ventilation, and then decreased gradually (P 0.05). After 12 hours of ventilation, the number of polymorphonuclear cells and the activity of neutrophil elastase increased significantly after 24 h ventilation. With the increase of PEEP, the expression of MUC5AC mRNA, the number of polymorphonuclear granulocytes and the activity of NE increased with the increase of TNF- 伪 IL-8 mRNA and protein content. Mechanical distraction could significantly increase the concentration of Ca~(2 in the epithelial cells of the human duct, increase the expression of SNAP23 and MUC5AC in the epithelial cells of the human duct, and increase the secretion of MUC5AC in the supernatant of the cell culture. The ED-LNA of nifedipine rcks could inhibit the expression of SNAP23 and MUC5AC and increase the secretion of MUC5AC by mechanical stretch (all P0.05%), but low molecular weight heparin could not significantly decrease the expression of SNAP23 and MUC5AC. The secretion of P0.05, U0126 and SB203580 inhibited the increase of MUC5AC mRNA and protein expression induced by distraction (both P0.01a). Conclusion Mechanical ventilation and stretch can promote the expression of MUC5AC mRNA and the secretion of MUC5AC in airway epithelial cells, which may be related to the inflammation induced by mechanical ventilation and stretch. Tension-sensitive cationic channel (Cam 2) is related to the MARCKS pathway and ERK1 / 2 p38 MAPK signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363.1

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