脂筏-神经酰胺在TNF-α激活人气道上皮细胞DUOX1酶中的作用

发布时间：2018-03-18 14:44

本文选题：脂筏　切入点：双重氧化酶-1(DUOX1)　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：呼吸道疾病如变应性鼻炎、哮喘等都是常见的变态反应性疾病。据研究发现,在其发病机制中,气道高反应性是其共同的病理生理特征。当气道上皮细胞受到体内外炎症因素刺激时,会通过气道上皮的氧化酶产生大量的活性氧,引起气道上皮的损伤进而导致气道高反应性引起疾病的发生。双重氧化酶-1(Dual oxidase-1, DUOX1)是气道上皮细胞产生活性氧的重要的氧化酶之一,在内外炎症因素的刺激下,它能够催化相应底物产生超氧阴离子并且还有超氧化物岐化酶(Superoxide dismutase, SOD)的作用将超氧阴离子歧化为过氧化氢(H_2O_2)。研究证明其在气道上皮活性氧产生中起着主要的作用。脂筏是细胞脂质双层内含有特殊脂质和蛋白质的膜微区,其有大量的胆固醇和鞘磷脂组成。当细胞受到炎症因素刺激时,脂筏中的鞘磷脂能在酸性鞘磷脂酶的作用下发生水解,释放出大量的神经酰胺(Ceramide),后者具有自发聚集的性质,其能够将细胞膜上面的小的膜微区聚集形成大的平台,氧化酶等生物活性大分子可以在这个平台聚集形成酶复合体而被激活并且相互作用,从而实现信号的跨膜转导。本研究拟探讨脂筏在肿瘤坏死因子α(Tumor necrosis factor-α, TNF-α)引起的DUOX1酶和其在胞浆中的调节亚基P47phox激活以及引起气道上皮损伤的信号转导机制中是否发挥作用,以期为变应性鼻炎、慢性支气管炎、哮喘等气道炎症性疾病的防治提供新的策略。目的:探讨脂筏-神经酰胺在TNF-α激活人气道上皮细胞DUOX1酶中的作用。方法:1.利用蔗糖超速离心法分离提取脂筏并用Western blot检测脂筏提取物中DUOX1和P47~(phox)的表达。2.利用激光共聚焦方法观察细胞胞膜上的DUOX1和P47~(phox)的表达,同时观察两者分别和脂筏标记蛋白霍乱毒素B亚单位(Cholera toxin B subunit, CTXB)和神经酰胺(Ceramide)的共定位现象。3.用活性氧检测试剂盒检测细胞内活性氧的生成。结果:在脂筏提取物中,TNF-α刺激细胞引起DUOX1和P47phox在细胞膜脂筏区中的表达增加(P0.05),脂筏干扰剂可以抑制TNF-α刺激引起的DUOX1和P47phox在细胞膜脂筏区中表达增加(P0.05);TNF-α刺激细胞后,在共聚焦显微镜下可以观察到DUOX1和P47phox在细胞膜上明显发生簇聚现象,同时DUOX1和P47phox分别与脂筏的标记物霍乱毒素和神经酰胺(Ceramide)呈现共定位现象,脂筏干扰剂可以抑制TNF-α刺激引起的DUOX1和P47phox在细胞膜上簇聚以及共定位现象;TNF-α刺激气道上皮细胞可以明显增加细胞内的活性氧生成(P0.05),脂筏的干扰剂以及DUOX1的抑制剂可以抑制TNF-α刺激引起的活性氧增加(P0.05)。结论:TNF-α可以引起气道上皮细胞内DUOX1、P47phox在脂筏中的含量增加,即DUOX1、P47phox向脂筏区转移,从而导致该酶的激活,刺激活性氧产生增加;酸性鞘磷脂酶抑制剂地昔帕明可以抑制上述过程,说明这一过程可能依赖于脂筏来源的神经酰胺。
[Abstract]:Respiratory diseases such as allergic rhinitis and asthma are common allergic diseases. Airway hyperresponsiveness is a common pathophysiological feature. When airway epithelial cells are stimulated by inflammatory factors in vivo and in vitro, a large amount of reactive oxygen species is produced through the oxidase of airway epithelium. Double Oxidase 1 (DUOX1) is one of the most important oxidases for the production of reactive oxygen species in airway epithelial cells, which is stimulated by internal and external inflammatory factors. It can catalyze the production of superoxide anion from the corresponding substrate and has the effect of superoxide dismutase (SOD) to dissect the superoxide anion into H _ 2O _ 2 H _ 2O _ 2. It has been proved that the superoxide anion plays a major role in the production of reactive oxygen species in airway epithelium. Lipid rafts are membrane microregions containing special lipids and proteins in the bilayer of cell lipids, which contain large amounts of cholesterol and sphingomyelin. When cells are stimulated by inflammatory factors, Sphingolipids in lipid rafts can be hydrolyzed by acidic sphingomyelinase, releasing a large amount of ceramide Ceramide-which has the property of spontaneous aggregation, and can aggregate the small membrane microregions above the membrane into a large platform. Bioactive macromolecules, such as oxidase, can be activated and interact with each other in this platform to form enzyme complexes, thus transmembrane transduction of signal can be realized. The aim of this study was to investigate the role of lipid rafts in the activation of tumor necrosis factor 伪 tumor necrosis factor- 伪 (TNF- 伪) -induced DUOX1 enzyme and its regulatory subunit P47phox in the cytoplasm, and in the signal transduction mechanism of airway epithelial injury, in order to induce allergic rhinitis. Prevention and treatment of airway inflammatory diseases such as chronic bronchitis and asthma provide new strategies. Aim: to investigate the role of lipid raft-ceramide in activation of DUOX1 enzyme in human epithelial cells by TNF- 伪. Methods 1. Extract lipid raft by sucrose ultracentrifugation and detect the expression of DUOX1 and P47phox in the extract of lipid raft by Western blot. The expression of DUOX1 and P47phox on cell membrane was observed by confocal laser scanning. The co-localization of Cholera toxin B subunit (CTXB) and ceramide (ceramide) were observed, respectively. Reactive oxygen species (Ros) detection kit was used to detect the production of reactive oxygen species (Ros) in the cells. Results: TNF- 伪 stimulated the expression of DUOX1 and P47phox in the lipid raft cell membrane. The lipid raft interference could inhibit the expression of DUOX1 and P47phox in the lipid raft region stimulated by TNF- 伪 and increase the expression of DUOX1 and P47phox in the lipid raft. Clusters of DUOX1 and P47phox on cell membrane were observed under confocal microscope, and DUOX1 and P47phox co-located with cholerae toxin and ceramide (ceramide), markers of lipid raft, respectively. Lipid raft interference could inhibit DUOX1 and P47phox aggregation and co-localization induced by TNF- 伪 stimulation. TNF- 伪 stimulated airway epithelial cells could significantly increase the production of reactive oxygen species (Ros) (P0.05), lipid raft disruptors and inhibitors of DUOX1. It could inhibit the increase of reactive oxygen species (Ros) induced by TNF- 伪. Conclusion: TNF- 伪 can increase the content of DUOX1P47phox in airway epithelial cells, that is, DUOX1P47phox transfer to lipid raft, which leads to the activation of DUOX1P47phox, and stimulates the production of reactive oxygen, and the acid sphingolipase inhibitor, desopramine, can inhibit the above process. It is suggested that this process may be dependent on ceramide derived from lipid rafts.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R765.21;R562.25

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