本文選題：脂筏　切入點：雙重氧化酶-1(DUOX1)　出處：《華中科技大學》2010年碩士論文　論文類型：學位論文

【摘要】：呼吸道疾病如變應性鼻炎、哮喘等都是常見的變態(tài)反應性疾病。據研究發(fā)現(xiàn),在其發(fā)病機制中,氣道高反應性是其共同的病理生理特征。當氣道上皮細胞受到體內外炎癥因素刺激時,會通過氣道上皮的氧化酶產生大量的活性氧,引起氣道上皮的損傷進而導致氣道高反應性引起疾病的發(fā)生。雙重氧化酶-1(Dual oxidase-1, DUOX1)是氣道上皮細胞產生活性氧的重要的氧化酶之一,在內外炎癥因素的刺激下,它能夠催化相應底物產生超氧陰離子并且還有超氧化物岐化酶(Superoxide dismutase, SOD)的作用將超氧陰離子歧化為過氧化氫(H_2O_2)。研究證明其在氣道上皮活性氧產生中起著主要的作用。 脂筏是細胞脂質雙層內含有特殊脂質和蛋白質的膜微區(qū),其有大量的膽固醇和鞘磷脂組成。當細胞受到炎癥因素刺激時,脂筏中的鞘磷脂能在酸性鞘磷脂酶的作用下發(fā)生水解,釋放出大量的神經酰胺(Ceramide),后者具有自發(fā)聚集的性質,其能夠將細胞膜上面的小的膜微區(qū)聚集形成大的平臺,氧化酶等生物活性大分子可以在這個平臺聚集形成酶復合體而被激活并且相互作用,從而實現(xiàn)信號的跨膜轉導。 本研究擬探討脂筏在腫瘤壞死因子α(Tumor necrosis factor-α, TNF-α)引起的DUOX1酶和其在胞漿中的調節(jié)亞基P47phox激活以及引起氣道上皮損傷的信號轉導機制中是否發(fā)揮作用,以期為變應性鼻炎、慢性支氣管炎、哮喘等氣道炎癥性疾病的防治提供新的策略。 目的:探討脂筏-神經酰胺在TNF-α激活人氣道上皮細胞DUOX1酶中的作用。 方法:1.利用蔗糖超速離心法分離提取脂筏并用Western blot檢測脂筏提取物中DUOX1和P47~(phox)的表達。2.利用激光共聚焦方法觀察細胞胞膜上的DUOX1和P47~(phox)的表達,同時觀察兩者分別和脂筏標記蛋白霍亂毒素B亞單位(Cholera toxin B subunit, CTXB)和神經酰胺(Ceramide)的共定位現(xiàn)象。3.用活性氧檢測試劑盒檢測細胞內活性氧的生成。 結果:在脂筏提取物中,TNF-α刺激細胞引起DUOX1和P47phox在細胞膜脂筏區(qū)中的表達增加(P0.05),脂筏干擾劑可以抑制TNF-α刺激引起的DUOX1和P47phox在細胞膜脂筏區(qū)中表達增加(P0.05);TNF-α刺激細胞后,在共聚焦顯微鏡下可以觀察到DUOX1和P47phox在細胞膜上明顯發(fā)生簇聚現(xiàn)象,同時DUOX1和P47phox分別與脂筏的標記物霍亂毒素和神經酰胺(Ceramide)呈現(xiàn)共定位現(xiàn)象,脂筏干擾劑可以抑制TNF-α刺激引起的DUOX1和P47phox在細胞膜上簇聚以及共定位現(xiàn)象;TNF-α刺激氣道上皮細胞可以明顯增加細胞內的活性氧生成(P0.05),脂筏的干擾劑以及DUOX1的抑制劑可以抑制TNF-α刺激引起的活性氧增加(P0.05)。 結論:TNF-α可以引起氣道上皮細胞內DUOX1、P47phox在脂筏中的含量增加,即DUOX1、P47phox向脂筏區(qū)轉移,從而導致該酶的激活,刺激活性氧產生增加;酸性鞘磷脂酶抑制劑地昔帕明可以抑制上述過程,說明這一過程可能依賴于脂筏來源的神經酰胺。
[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 偽 tumor necrosis factor- 偽 (TNF- 偽) -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- 偽. 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- 偽 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- 偽 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- 偽 stimulation. TNF- 偽 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- 偽. Conclusion: TNF- 偽 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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R765.21;R562.25

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