本文選題：阿薩希毛孢子菌　切入點(diǎn)：生物膜　出處：《山西醫(yī)科大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 研究背景 毛孢子菌屬于半知菌亞門(mén),芽生菌綱,隱球酵母目,隱球酵母科。在自然界中廣泛存在,是口腔、皮膚、指甲的正常寄生菌。它可引起淺部感染,如毛發(fā)、指甲、皮膚的白毛結(jié)節(jié)病和免疫缺陷患者的系統(tǒng)性或播散性感染,主要見(jiàn)于惡性腫瘤、血液病、呼吸功能不全、慢性腎功能不全、糖尿病、肝硬化或艾滋病患者。其主要風(fēng)險(xiǎn)因素有：抗生素、腎上腺皮質(zhì)激素、免疫抑制劑、細(xì)胞毒性藥物的使用,和各種醫(yī)療設(shè)備的應(yīng)用如機(jī)械通氣、導(dǎo)尿術(shù)、靜脈插管、移植、手術(shù)操作或持續(xù)性腹膜透析等。目前尚有關(guān)于健康者感染此病原菌導(dǎo)致系統(tǒng)性感染的報(bào)告。阿薩希毛孢子菌是其重要臨床致病菌。 阿薩希毛孢子菌(asahii trichosporon,T. asahii)所致播散性毛孢子菌病的臨床研究中發(fā)現(xiàn),盡管使用抗真菌藥物,但感染仍持續(xù)存在。2006年Giovanni等發(fā)現(xiàn)與浮游狀態(tài)的菌株相比,阿薩希毛孢子菌生物膜細(xì)胞對(duì)兩性霉素B、卡泊芬凈、伏立康唑和氟康唑的最低抑菌濃度(MICs)明顯增加,國(guó)內(nèi)李繼紅等也發(fā)現(xiàn)固著相即生物膜細(xì)胞的MIC比浮游相成倍提高。導(dǎo)致耐藥性增加的原因是什么? 研究目的 1本課題首先模擬體內(nèi)環(huán)境在常用醫(yī)學(xué)材料表面構(gòu)建阿薩希毛孢子菌(T. asahii)生物膜(biofilm,BF)模型,證明其存在并在倒置顯微鏡及掃描電鏡下觀察其超微結(jié)構(gòu)形態(tài)特征,從形態(tài)結(jié)構(gòu)方面探索生物膜耐藥的機(jī)理。 2采用FDA/PI雙熒光染色和激光共聚焦顯微鏡相結(jié)合的技術(shù)以2μm為步距觀察阿薩希毛孢子菌生物膜在不同時(shí)間菌的分布情況和成熟期不同層面生物膜菌分布和活性,分析其耐藥機(jī)制。 3初步觀察不同材料對(duì)阿薩希毛孢子菌(T. asahii)生物膜(biofilm,BF)形成能力的影響。 研究方法 1以來(lái)源于本院皮膚科臨床分離株阿薩希毛孢子菌株(BZP07002)1株為研究對(duì)象,在聚芳脂材料上構(gòu)建T. asahii生物膜模型,不同時(shí)間觀察其在倒置顯微鏡和掃描電鏡下的形態(tài)特征,并同時(shí)用XTT法和細(xì)胞計(jì)數(shù)法測(cè)定生物膜活性進(jìn)行定量分析。 2激光共聚焦顯微鏡下觀察經(jīng)FDA/PI染色生物膜不同時(shí)間菌分布和成熟期不同層面菌分布、活性及生物膜厚度。 3將聚芳脂(PAT)、聚苯乙烯(PS)、聚氯乙烯(PVC)材料剪成1×1cm2大小,放入24孔板中構(gòu)建T. asahii生物膜模型,采用XTT法測(cè)定生物膜活性進(jìn)行定量分析,不同時(shí)間觀察T. asahii生物膜在倒置顯微鏡下的形態(tài)特征,將聚芳脂、聚苯乙烯上培養(yǎng)72h的生物膜在掃描電鏡下觀察。 結(jié)果 1.T. asahii可在聚芳脂表面形成生物膜,其形成過(guò)程包括了真菌表面粘附、微菌落形成和生物膜成熟；鏡下T. asahii生物膜的孢子、菌絲、假菌絲等多種形態(tài)相互堆積纏繞,與基質(zhì)共同形成一個(gè)復(fù)雜的三維立體結(jié)構(gòu)。隨時(shí)間延長(zhǎng),生物膜活性不斷升高,XTT法和活菌計(jì)數(shù)結(jié)果呈正相關(guān)(r=0.936,P0.001)； 2.隨時(shí)間延長(zhǎng),阿薩希毛孢子菌逐漸由散在粘附到聚集成團(tuán)塊,活菌和死菌的量都增加；T. asahii生物膜不同層面的活性曲線(xiàn)坡度緩和,無(wú)明顯規(guī)律,生物膜的厚度為14.3μm到31μm不等。 3.聚芳脂、聚苯乙烯、聚氯乙烯3種材料上均能形成生物膜,且形成的生物膜廣泛,幾乎覆蓋整個(gè)材料表面,不同材料上形成生物膜的活性有差別(F=14.743,P0.01),48h時(shí)活性由高到低為聚芳脂=聚氯乙烯聚苯乙烯。倒置顯微鏡和掃描電鏡下觀察發(fā)現(xiàn)聚芳脂、聚氯乙烯形成的生物膜可見(jiàn)孢子、菌絲、假菌絲結(jié)構(gòu),聚苯乙烯上形成以孢子為主要結(jié)構(gòu)的微生物群落。 結(jié)論 1.體外可形成T. asahii生物膜；T. asahii生物膜比其浮游狀態(tài)下具有更加復(fù)雜的結(jié)構(gòu)； 2.隨時(shí)間延長(zhǎng)T. asahii生物膜活性不斷增高,不同層面T. asahii生物膜的活性無(wú)明顯差異,分析細(xì)胞低生長(zhǎng)率并非T. asahii生物膜的耐藥機(jī)制。3.T. asahii可在不同的材料上形成生物膜,但不同材料上形成生物膜的能力不同。聚芳脂、聚氯乙烯比聚苯乙烯更易于真菌的粘附；且以菌絲、假菌絲為主要結(jié)構(gòu)的微生物群落活力比單純孢子的活力強(qiáng)。
[Abstract]:Research background
Spore bacteria belong to deuteromycotina, blastomycete Gang, Cryptococcus yeast, yeast Cryptococcus. Exist widely in nature is the mouth, skin, normal parasite nails. It can cause superficial infections, such as hair, nails, skin white haired patients with sarcoidosis and immunodeficiency of systemic or disseminated infection, mainly in malignant tumor, blood disease, respiratory insufficiency, chronic renal insufficiency, diabetes, cirrhosis or AIDS patients. The main risk factors are: antibiotics, glucocorticoids, immunosuppressants, use of cytotoxic drugs, and other medical equipment applications such as mechanical ventilation, venous catheterization. Intubation, transplantation, surgical operation or continuous peritoneal dialysis. There are about to lead to systemic infection infection Report of the pathogen and health. ASA Trichosporon is one of the most important clinical pathogens.
A Csathy Mao (asahii Trichosporon, T. spores asahii) found in clinical research due to disseminated trichosporosis in, despite the use of antifungal drugs, but the infection persists in.2006 Giovanni found with planktonic strains and compared to ASA spore bacteria biofilm cells of amphotericin B, caspofungin, MIC voriconazole and fluconazole (MICs) increased significantly, domestic Li Jihong also found that phase sessile biofilm cells MIC times higher than planktonic phase. What is the cause of the increase of drug resistance?
research objective
1 this paper simulated in vivo environment in the construction of ASA and Trichosporon surface (T. asahii) commonly used in medical materials biofilm (biofilm, BF) model, and prove the existence and to observe the ultrastructure morphological features in the inverted microscope and scanning electron microscope, the mechanism of biofilm resistance from the cable structure.
2 by FDA/PI double fluorescent staining and laser confocal microscope technology combined with 2 m to step away from the observation that ASA spore bacteria biofilm distribution in different time and different levels of bacteria mature biofilm bacteria distribution and activity analysis of its drug resistance mechanism.
3 the effects of different materials on the formation ability of T. asahii (T. asahii) biofilm (biofilm, BF) were preliminarily observed.
research method
Since 1 from the Department of dermatology in our hospital clinical isolates of ASA and Trichosporon strains 1 strains (BZP07002) as the research object, construct the T. asahii biofilm model in aromatic lipid materials, different time were observed under inverted microscope and scanning electron microscope morphological characteristics, and at the same time by XTT method and cell counting method for the determination of biological membrane the activity of quantitative analysis.
Under 2 laser confocal microscopy, the distribution of bacteria in different layers of the biofilm and the distribution of bacteria, activity and the thickness of the biofilm were observed at different time of FDA/PI staining.
3 aromatic lipid (PAT), polystyrene (PS), polyvinyl chloride (PVC) material is cut into 1 x 1cm2 size, into 24 well plates to construct T. asahii biofilm model, determination of biological activity were quantitatively analyzed by XTT method, different time to observe T. asahii biofilm morphology under inverted microscope, the poly aryl fat, polystyrene biofilm 72h under scanning electron microscope.
Result
1.T. asahii can form biofilms in aromatic lipid surface, its formation process consists of fungal adherence, microcolony formation and mature biofilm; microscopic T. asahii biofilm spores, mycelium, various forms of pseudohypha and stacking winding, together form a complex three-dimensional structure and matrix. With the extension of time biological activity, increasing XTT method and viable count were positively correlated (r=0.936, P0.001);
2., with the extension of time, the amount of viable and dead bacteria increased gradually from sporadic adherence to aggregated agglomerates, and the slope of T. asahii biofilms varied at different levels without obvious regularity. The thickness of biofilms ranged from 14.3 m to 31 m.
3. aromatic resin, polystyrene, polyvinyl chloride 3 materials can form biofilm, biofilm formation and wide, covering almost the entire surface of the material, the formation of biofilm on different materials have different activity (F=14.743, P0.01), 48h activity from high to low fat = poly aryl polyvinyl chloride polystyrene. Found polyaryl inverted microscope and scanning electron microscope observation of lipid, biofilm formation of visible spores, hyphae and pseudohyphae of PVC, structure, formation of the microbial community with spores for the main structure of polystyrene.
conclusion
1. the T. asahii biofilm can be formed in vitro, and the T. asahii biofilm has a more complex structure than its floating state.
2. with the prolonging of T. asahii biofilm activity increased, no significant difference between the different levels of T. asahii biofilm activity, analysis of cell growth rate is not low T. asahii biofilm resistance mechanism of.3.T. asahii biofilm formation in different materials, but the ability of biofilm formation in different materials. Different aromatic grease the adhesion of PVC more easily than polystyrene and fungi; mycelium, pseudohypha activity of microbial community as the main structure than single spore viability.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R379

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