新生隐球菌生物膜外分泌蛋白初步分析的研究

发布时间：2018-01-13 14:25

本文关键词：新生隐球菌生物膜外分泌蛋白初步分析的研究　出处：《第二军医大学》2012年硕士论文　论文类型：学位论文

更多相关文章： 新生隐球菌 生物膜 MTT减低法 iTRAQ

【摘要】：生物膜是指由细胞外多聚基质以及被粘结的菌团组成。换言之，生物膜是一种附着于活组织或无活力组织的表面、由其自身产生的ECM包裹的由结构的菌细胞群体，是一种独特而复杂、相互依存的与表面有关的微生物群体，是细菌、真菌在生长过程中为适应生存环境而形成的一种与悬浮细胞相对应的生存方式。生物膜的显著特点是具有高度耐药性。目前估计约65％的人类感染与生物膜有关[1]，而有学者认为大于80％的人类感染中有生物膜的形成[2]。近30年来随着广谱抗生素和免疫抑制剂的大量使用，加上癌症及器官移植患者的增加，以及医学材料使用的增多，新生隐球菌感染呈上升之势。其中相当一部分与新生隐球菌形成生物膜密切相关。感染部位一旦形成生物膜或植入人体内的生物材料表面一旦形成了生物膜，由于其难以被大多数抗真菌药物等彻底杀灭，新生隐球菌生物膜引起的感性性疾病因而大都是慢性和难治性的。近年来有关新生隐球菌生物膜的基础研究和临床研究已引起国内外学者的高度重视。为了深入了解新生隐球菌生物膜的相关特征，首先应该对其形成的生物膜进行形态学观察。研究表明，，生物膜的形成可以有几个明显的阶段；MTT比色法是目前定量研究真菌生物膜最常用的方法，能够间接反映生物膜中活细胞的数量，从而分析生物膜的形成过程。iTRAQ技术在分离蛋白质混合样品，比较差异方面有不可替代的作用，它是目前比较新的一种分离成千上万个蛋白质组份的方法，与传统的双向电泳技术相比，在差异蛋白鉴定种类、分子量、灵敏性等方面都有着不可比拟的优势。本研究利用倒置相差显微镜及MTT比色法分析新生隐球菌生物膜形成的动态过程，为进一步了解新生隐球菌生物膜特性及其耐药机制提供理论依据；同时采用基础培养基96孔板培养的方法构建生物膜体外模型以及将菌悬液加入细胞培养瓶置于摇床上低速培养的方法构建新生隐球菌悬浮态模型，提取上清液中的外分泌蛋白，对外分泌蛋白进行初步纯化处理后运用iTRAQ技术对其进行初步分析，寻找与生物膜形成有关的蛋白分子，为进一步了解新生隐球菌群体效应提供理论基础。综上所述，新生隐球菌可以在微孔板上形成典型的、成熟的生物膜结构，其形成过程有早期、中期、晚期3个阶段。新生隐球菌生物膜活性随培养时间延长而增加，48h后代谢活性相对稳定。结论：新生隐球菌生物膜具有三维立体空间结构，其结构具有多样、不均质、开放的特点。同时运用iTRAQ技术初步分析：可看到差异蛋白存在。
[Abstract]:Biofilm is made up of extracellular polymeric matrix and bound bacteria. In other words, biofilm is a kind of surface attached to living or inactive tissue. The ECM encapsulated by the structure of the bacterial cell population, is a unique and complex, interdependent and surface related microbial population, is a bacterium. A form of survival corresponding to suspended cells formed during the growth of fungi to adapt to the living environment. Biofilms are characterized by high drug resistance. It is currently estimated that about 65% of human infections and biofilms have. Pass. [1, and some scholars believe that biofilms are formed in human infections greater than 80%. [2]. In the last 30 years, with the extensive use of broad-spectrum antibiotics and immunosuppressants, the number of cancer and organ transplant patients has increased, and the use of medical materials has increased. Cryptococcus neoformans infection is on the rise. Many of them are closely related to the formation of biofilm of Cryptococcus neoformans. Once the biofilm is formed in the infected site or the biomaterial surface implanted into human body, biofilm is formed. Because it is difficult to be killed thoroughly by most antifungal drugs and so on. Most of the sensitive diseases caused by Cryptococcus neoformans biofilm are chronic and refractory. In recent years, the basic and clinical studies on Cryptococcus neoformans biofilm have attracted great attention from scholars at home and abroad. In order to understand the characteristics of Cryptococcus neoformans biofilm, it is necessary to observe the morphology of the biofilm formed by Cryptococcus neoformans. MTT colorimetry is the most commonly used method for quantitative study of fungal biofilm, which can indirectly reflect the number of living cells in biofilm. In order to analyze the biofilm formation process. ITRAQ technology in the separation of mixed protein samples, compare the difference has an irreplaceable role. It is a relatively new method for the separation of thousands of protein components, compared with the traditional two-dimensional electrophoresis technology, in differential protein identification, molecular weight. In this study the dynamic process of biofilm formation of Cryptococcus neoformans was analyzed by inverted phase contrast microscope and MTT colorimetry. To further understand the characteristics of Cryptococcus neoformans biofilm and drug resistance mechanism to provide a theoretical basis; At the same time, the biofilm model was established by 96 hole plate culture of basic culture medium and the suspension model of Cryptococcus neoformans was constructed by adding bacteria suspension into cell culture bottle and low speed culture on shaking bed. The exocrine proteins were extracted from supernatant and purified and analyzed by iTRAQ technique to find the protein molecules related to the formation of biofilm. It provides a theoretical basis for further understanding the colony effect of Cryptococcus neoformans. To sum up Cryptococcus neoformans can form a typical mature biofilm structure on the micropore plate. The formation of Cryptococcus neoformans takes place in the early and middle stages. The biofilm activity of Cryptococcus neoformans was relatively stable after 48 h with the increase of culture time. Conclusion: Cryptococcus neoformans biofilm has three-dimensional spatial structure. Its structure is diverse, uneven and open. At the same time, using iTRAQ technique, we can see the existence of differential protein.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R379

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