【作者】 张慧；

【导师】 孙红英； 李继扬；

【作者基本信息】 复旦大学 ， 口腔临床医学（专业学位）， 2014， 硕士

【摘要】 目的：本实验拟通过体外构建白念珠菌生物膜的可重复模型,观察分析紫红素和氟康唑对白念珠菌生物膜形态结构及线粒体的影响,并从基因角度检测黏附相关基因ALS1、ALS4、EFG1和HWP1的表达水平变化,以期初步探索紫红素对白念珠菌生物膜黏附过程的抑制机制。方法：1.白念珠菌生物膜构建及鉴定：选择白念珠菌标准株ATCC90028体外构建生物膜模型,利用倒置相差显微镜、扫描电镜及激光共聚焦显微镜观察生物膜的形态结构,并通过XTT减低法测定白念珠菌生物膜的动力生长曲线,明确生长对数期及衰亡期。2.紫红素和氟康唑对白念珠菌生物膜形成过程的影响：通过XTT减低法和细胞毒性实验确定紫红素的最佳作用浓度,并通过倒置相差显微镜、扫描电镜及激光共聚焦显微镜三种方法观察药物作用后生物膜的结构变化情况。3.紫红素和氟康唑对白念珠菌生物膜黏附过程的影响：倒置相差显微镜下观察紫红素和氟康唑对黏附过程的影响；利用称量于重的方法、基因检测ALS1、ALS4、EFG1和HWP1表达水平的变化定量分析紫红素和氟康唑对黏附过程的影响,并通过激光共聚焦显微镜观察紫红素和氟康唑对生物膜线粒体的影响,初步探索黏附作用机制。结果：1.白念珠菌生物膜的体外模型稳定且可重复性高,镜下生物膜呈典型的网状结构,0.01%结晶紫染色后可见菌丝结构及胞外基质。白念珠菌生物膜的动力生长曲线基本呈“S”型,对数生长期大约为12～48h,稳定期为48～60h为,60h后开始进入衰亡期。2.XTT减低法和细胞毒性实验确定紫红素最佳作用浓度为20gg/mL,该浓度既能达到良好的抑制作用,又不致于对人体正常成纤维细胞造成伤害；倒置相差显微镜、扫描电镜及激光共聚焦显微镜下可观察到不同浓度紫红素和氟康唑作用后生物膜的形态,20μg/mL紫红素作用后的生物膜不能形成正常的菌丝且结构松散、胞外基质几乎没有。3.倒置相差显微镜下20μg/mL紫红素作用2h的生物膜细胞量少于128μg/mL氟康唑组及正常对照组；作用48后,经消化离心、干燥后的生物膜重量,同样是20μg/mL紫红素组干重量最低,与其余两组相比P值均小于0.05；基因ALS1、ALS4、EFG1和HWP1的表达量却高于128μg/mL氟康唑组,低于正常对照组,两两比较所得P值均小于0.05；激光共聚焦显微镜下,紫红素作用后生物膜中线粒体的活性明显减弱。结论：1.白念珠菌标准株ATCC90028可在体外形成生物膜结构,且所构建的生物膜模型稳定性好、重复性高。2.与氟康唑组和正常对照组相比,紫红素对白念珠菌生物膜的形成具有明显地抑制作用。3.与氟康唑组和正常对照组相比,紫红素对白念珠菌生物膜的黏附过程具有明显的抑制作用,除通过抑制黏附相关基因ALS1、ALS4、EFG11和HWP1的表达水平,尚通过线粒体相关的其他途径发挥抑制作用。更多还原

【Abstract】 Objectives:This study was to construct Candida albicans biofilms model in vitro which can be repeated. Objecting and analyzing the effect on biofilms structure and mitochondria by purpurin and fluconazole, and detecting the expression level of adhesion genes ALS1, ALS4, EFG1, HWP1 to the preliminary exploration of mechanism about the adhesion process of Candida albicans under purpurin.Methods:1. Constrution and identification of Candida albicans biofilms:Slecting the standard strains of Candida albicans ATCC90028 to forming the biofilms in vitro, and observing the morphological structure using the inverted phase contrast microscope, scanning electron microscope and confocal microscopy. Using the XTT reduction technique to measure the growth of Candida albicans bioflms, especially to determine the logarithmic growth phase and declining phase.2. The impact of purpurin and fluconazole against formation of Candida albicans biofilms:To make sure the optimal concentrion of purpurin against Candida albicnas biofilms, we used the XTT reduction assays and cytotoxicity experiments. And also using the inverted phase contrast microscope, scanning electron microscope and confocal microscopy to observe the morphological structure of treated biofilms with purpurin and fluconazole.3. The impact of purpurin and fluconazole against Candida albicans biofilms adhesion process:Observing the effect of adhesion process with purpurin and fluconazole via the inverted phase contrast microscope, the activity of mitochondria via confocal microscopy in a qualitative method. By way of weighing the dry weight and detecting the expression level of gene ALS1, ALS4, EFG1, HWP1 in a quantitive method to exploring the mechanism about the adhesion.Results:1. The model of biofilms was stable and reproducible, microscopic biofilm was a tipical network structure, the hyphae structure and extracellular matrix were more apparent after staining with 0.01% crystal violet. The growth curve was nearly a "S" shape, and the logarithmic phase was about 12-48h, stable period was about 48-60h, after the 60h entering into the decling phase.2. The final concentration of purpurin was 20μg/mL by XTT reduction assays and cytotoxicity experiments, the concentration had an effective consequent and no toxicity on human fibrolast cells. Under the inverted phase contrast microscope, scanning electron microscope and confocal microscopy, the biofilms teated with different concentrations of purpurin and fluconazole were observed. And the biofilms were more loose and the hyphae structure were less than any other groups.3. Under the inverted phase contrast microscope, with 20μg/mL purpurin biofilm cells were less than any other groups. Incubated for 48h, after digestion and centrifugation, the weight of biofilms were lowest under 20μg/mL purpurin, comparing with other two groups the P values were less than 0.05. However, the genes expression levels were higher than the fluconazole groups, lower than normal groups. And the P values were less than 0.05. Finally, the activity of mitochondrial were significantly reduced with 20μg/mL purpunn.Conclusions:1. Standard strains of Candida albicans ATCC90028 could form the stable and repeatable biofilm model in vitro.2. Compared with fluconazole group and normal group, purpurin groups had a significantly inhibition on biofilms.3. Compared with fluconazole group and normal group, purpurin groups had a significantly inhibition on adhension process about biofilms.更多还原