气道粘蛋白大分子末端唾液酸化对粘蛋白—铜绿假单胞菌相互作用的影响及其机制

发布时间：2018-04-02 04:29

本文选题：气道　切入点：粘蛋白　出处：《第三军医大学》2006年博士论文

【摘要】： 目的: 气道急性和慢性炎症过程中粘液及粘蛋白高分泌,糖基化程度也显著增加。唾液酸作为主要的末端糖基之一,也是多种细菌可结合的糖受体,其增多与感染严重程度密切相关。而COPD等气道慢性炎症还存在着分泌细胞增生肥大等可能易于发生高分泌及高度唾液酸化的病理学基础。这些改变的意义值得深入研究。本课题拟:(1)比较COPD与肺炎患者气道粘蛋白大分子末端唾液酸化的差异;(2)体外干预其末端唾液酸,观察对粘蛋白-铜绿假单胞菌相互作用的影响及两组标本相应变化的异同;(3)比较无明显感染的COPD和肺功能正常的对照组患者支气管粘膜中常见唾液酸转移酶mRNA表达水平的差异性;(4)体内应用相关干预因素,观察对盐酸致伤大鼠气管内接种细菌的影响。旨在探讨糖类抗细菌粘附剂的抗感染作用效果及机制,为进一步的药物研发提供实验依据。方法: 1、收集24例COPD和无COPD及其他慢性呼吸系统疾病的肺炎患者(以下分别简称COPD组和肺炎组)气管插管或切开后的气道吸引物,用分次离心和琼脂糖CL-2B凝胶层析的方法分离纯化粘蛋白大分子;琼脂糖凝胶电泳及免疫印迹验证分离效果,试剂盒测定总唾液酸含量,凝集素ELLA法分别比较唾液酸α2,3-和α2,6-结构含量; 2、粘蛋白样品包被在硅化膜片上,分别用弱碱+硼氢化钠做β-消去、特异性植物凝集素Maackia amurensis agglutinin(MAA)和Sambucus nigra agglutinin(SNA)、α2,3-特异性唾液酸酶和非特异性唾液酸酶处理,然后接种铜绿假单胞菌,或者是唾液酸双糖NeuAcα2-3Gal和NeuAcα2-6Gal分别与细菌混合接种,30min后解离细菌,定量培养以测定对细菌粘附的抑制作用;LB液体培养基中分别加入唾液酸双糖、特异性凝集素,以及α2,3-特异性唾液酸酶和非特异性唾液酸酶,混入菌液后加到粘蛋白包被的膜片上,7d后扫描电镜判断细菌生物被膜是否形成,解离细菌、定量培养测定膜片上的活菌数,以分析各种因素的抑制作用;
[Abstract]:Objective:Mucin and mucin are highly secreted and glycosylation is significantly increased during acute and chronic airway inflammation.Sialic acid, as one of the main terminal glycosyl groups, is also the binding sugar receptor of many bacteria. The increase of sialic acid is closely related to the severity of infection.However, chronic airway inflammation such as COPD also has the pathological basis of secretory cell proliferation and hypertrophy, which may be prone to hypersecretion and high saliva acidification.The significance of these changes deserves further study.To compare the difference of salivary acidification between COPD and pneumonia patients with airway mucin macromolecule terminal sialic acid in vitro.Observation of the effect on the interaction between mucin and Pseudomonas aeruginosa and the difference of the corresponding changes between the two groups) mRNA table of sialic acid transferase in bronchial mucosa of patients without obvious infection and normal pulmonary function(4) use of relevant intervention factors in vivo,To observe the effect of bacteria inoculation in trachea of rats injured by hydrochloric acid.The aim of this study was to investigate the anti-infective effect and mechanism of carbohydrate anti-bacterial adhesives and to provide experimental evidence for further drug development.Methods:1. Tracheal intubation and tracheotomy were collected from 24 patients with COPD and pneumonia without COPD and other chronic respiratory diseases (COPD group and pneumonia group respectively).The macromolecules of mucin were separated and purified by fractionation centrifugation and agarose CL-2B gel chromatography, the separation effect was verified by agarose gel electrophoresis and Western blotting, and the total sialic acid content was determined by kit.Lectin ELLA method was used to compare the content of sialic acid 伪 _ 2O _ 3- and 伪 _ 2O _ 6-, respectively.2. The mucin samples were coated on the silicide membrane and treated with weak alkali sodium borohydride as 尾 -elimination, specific phytoagglutinin Maackia amurensis agglutininine MAA) and Sambucus nigra agglutinine SNAA, 伪 _ 2N _ 3-specific sialidase and nonspecific sialidase respectively, and then inoculated with Pseudomonas aeruginosa.Or sialic acid disaccharide NeuAc 伪 2-3Gal and NeuAc 伪 2-6Gal were inoculated with bacteria for 30 minutes, then the bacteria were dissociated. The inhibitory effect of sialic acid disaccharide 2-3Gal and NeuAc 伪 2-6Gal on bacterial adhesion was determined by quantitative culture. The sialic acid disaccharide and specific lectin were added into LB liquid medium respectively.And 伪 _ 2o _ 3-specific sialidase and non-specific sialidase were added to the membrane coated with mucin for 7 days, then the bacterial biofilm was formed, the bacteria were dissociated, and the number of live bacteria on the membrane was determined by quantitative culture.To analyze the inhibitory effects of various factors;
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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