本文關(guān)鍵詞：新生隱球菌生物膜外分泌蛋白初步分析的研究　出處：《第二軍醫(yī)大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 新生隱球菌 生物膜 MTT減低法 iTRAQ

【摘要】：生物膜是指由細(xì)胞外多聚基質(zhì)以及被粘結(jié)的菌團(tuán)組成。換言之，生物膜是一種附著于活組織或無(wú)活力組織的表面、由其自身產(chǎn)生的ECM包裹的由結(jié)構(gòu)的菌細(xì)胞群體，是一種獨(dú)特而復(fù)雜、相互依存的與表面有關(guān)的微生物群體，是細(xì)菌、真菌在生長(zhǎng)過(guò)程中為適應(yīng)生存環(huán)境而形成的一種與懸浮細(xì)胞相對(duì)應(yīng)的生存方式。生物膜的顯著特點(diǎn)是具有高度耐藥性。目前估計(jì)約65％的人類(lèi)感染與生物膜有關(guān)[1]，而有學(xué)者認(rèn)為大于80％的人類(lèi)感染中有生物膜的形成[2]。近30年來(lái)隨著廣譜抗生素和免疫抑制劑的大量使用，加上癌癥及器官移植患者的增加，以及醫(yī)學(xué)材料使用的增多，新生隱球菌感染呈上升之勢(shì)。其中相當(dāng)一部分與新生隱球菌形成生物膜密切相關(guān)。感染部位一旦形成生物膜或植入人體內(nèi)的生物材料表面一旦形成了生物膜，由于其難以被大多數(shù)抗真菌藥物等徹底殺滅，新生隱球菌生物膜引起的感性性疾病因而大都是慢性和難治性的。近年來(lái)有關(guān)新生隱球菌生物膜的基礎(chǔ)研究和臨床研究已引起國(guó)內(nèi)外學(xué)者的高度重視。 為了深入了解新生隱球菌生物膜的相關(guān)特征，首先應(yīng)該對(duì)其形成的生物膜進(jìn)行形態(tài)學(xué)觀察。研究表明，，生物膜的形成可以有幾個(gè)明顯的階段；MTT比色法是目前定量研究真菌生物膜最常用的方法，能夠間接反映生物膜中活細(xì)胞的數(shù)量，從而分析生物膜的形成過(guò)程。iTRAQ技術(shù)在分離蛋白質(zhì)混合樣品，比較差異方面有不可替代的作用，它是目前比較新的一種分離成千上萬(wàn)個(gè)蛋白質(zhì)組份的方法，與傳統(tǒng)的雙向電泳技術(shù)相比，在差異蛋白鑒定種類(lèi)、分子量、靈敏性等方面都有著不可比擬的優(yōu)勢(shì)。本研究利用倒置相差顯微鏡及MTT比色法分析新生隱球菌生物膜形成的動(dòng)態(tài)過(guò)程，為進(jìn)一步了解新生隱球菌生物膜特性及其耐藥機(jī)制提供理論依據(jù)；同時(shí)采用基礎(chǔ)培養(yǎng)基96孔板培養(yǎng)的方法構(gòu)建生物膜體外模型以及將菌懸液加入細(xì)胞培養(yǎng)瓶置于搖床上低速培養(yǎng)的方法構(gòu)建新生隱球菌懸浮態(tài)模型，提取上清液中的外分泌蛋白，對(duì)外分泌蛋白進(jìn)行初步純化處理后運(yùn)用iTRAQ技術(shù)對(duì)其進(jìn)行初步分析，尋找與生物膜形成有關(guān)的蛋白分子，為進(jìn)一步了解新生隱球菌群體效應(yīng)提供理論基礎(chǔ)。 綜上所述，新生隱球菌可以在微孔板上形成典型的、成熟的生物膜結(jié)構(gòu)，其形成過(guò)程有早期、中期、晚期3個(gè)階段。新生隱球菌生物膜活性隨培養(yǎng)時(shí)間延長(zhǎng)而增加，48h后代謝活性相對(duì)穩(wěn)定。結(jié)論：新生隱球菌生物膜具有三維立體空間結(jié)構(gòu)，其結(jié)構(gòu)具有多樣、不均質(zhì)、開(kāi)放的特點(diǎn)。同時(shí)運(yùn)用iTRAQ技術(shù)初步分析：可看到差異蛋白存在。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R379

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