龙血素A抗白念珠菌生物被膜的作用及秀丽隐杆线虫感染模型的研究

发布时间：2018-01-09 15:31

本文关键词：龙血素A抗白念珠菌生物被膜的作用及秀丽隐杆线虫感染模型的研究　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：白念珠菌是存在于人的粘膜表面、胃肠道和泌尿生殖道中的条件致病真菌,当人的免疫系统受损时可导致严重感染的发生。许多白念珠菌感染是由于在植入的医疗装置上,如心脏支架、尿道插管等,形成生物被膜引起的。生物被膜导致反复感染和对常用的抗真菌药物的高度耐药,因此临床上亟需抗白念珠菌生物被膜的药物。抗真菌感染药物的研究需要动物模型,但是,常用的动物模型-小鼠感染模型成本高,实验周期长,不适用于大批量筛选药物,此外,也不适用于批量考察白念珠菌的关键致病因素。因此,抗真菌感染的研究需要高通量、低成本的体内感染模型。本课题开展了两部分的研究,第一部分研究了龙血素A抗白念珠菌生物被膜的作用,第二部分考察了秀丽隐杆线虫-白念珠菌感染模型在评价白念珠菌致病力和筛选抗感染药物方面的应用。在第一部分的研究中,我们用2,3-双-(2-甲氧基-4-硝基-5-磺苯基)-2H-四唑-5-甲酰苯胺法筛选抗白念珠菌生物被膜的药物时,发现龙血素A具有较好的活性。龙血素A是从血竭中分离的苯丙素药物,血竭是从龙血树中获得的树脂。在XTT法和扫描电子显微镜法的实验中,我们发现≥5μg/ml龙血素A对白念珠菌生物被膜有显著的抑制作用,而5-40μg/ml的龙血素A对白念珠菌没有杀菌和抑菌作用。在≥5μg/ml时,龙血素A能够显著降低细胞表面疏水性。在≥20μg/ml时,龙血素A抑制液体和固体菌丝诱导培养基中的菌丝形成。同时,Real Time RT-PCR结果表明龙血素A使粘附相关基因、菌丝相关基因和生物被膜相关基因的表达下调。此外,龙血素A在蜡螟感染模型和秀丽隐杆线虫液体感染模型中显示出体内抗感染作用,并表现出较低的毒性。总的来说,龙血素A表现出抗白念珠菌生物被膜的作用,并且这种作用可能与抑制粘附和菌丝形成相关。在第二部分的研究中,我们考察了秀丽隐杆线虫-白念珠菌体内感染模型,比较和评价了在液体培养基中和固体培养基上的秀丽隐杆线虫感染模型用于考察白念珠菌致病力和药物抗感染作用的优势和劣势。首先,我们成功地重现了秀丽隐杆线虫glp-4;sek-1液体感染模型,并用白念珠菌野生型SC5314、菌丝缺陷型hog1Δ/Δ、菌丝缺陷型pbs2Δ/Δ和菌丝缺陷型hst7Δ/Δ进行秀丽隐杆线虫glp-4;sek-1液体感染模型的实验证明该模型可以考察白念珠菌的致病力,发现白念珠菌菌丝缺陷型hog1Δ/Δ、pbs2Δ/Δ和hst7Δ/Δ感染组的生存率确实比野生型SC5314感染组的高;用抗真菌药物-氟康唑和抗白念珠菌生物被膜药物-龙血素A进行秀丽隐杆线虫glp-4;sek-1液体感染模型的实验证明该模型可以考察药物的的抗真菌作用,发现氟康唑和龙血素A药物治疗后确实提高了秀丽隐杆线虫的生存率;用免疫增强药物-黄芪进行秀丽隐杆线虫glp-4;sek-1液体感染模型的实验检测该模型是否可以考察药物的免疫调节作用,但是黄芪药物作用后没有提高秀丽隐杆线虫的生存率。即秀丽隐杆线虫glp-4;sek-1液体感染模型可用于考察白念珠菌的致病力和药物的抗真菌作用,但不能考察药物的免疫增强作用。因为秀丽隐杆线虫glp-4;sek-1免疫缺陷,不能用于研发增强免疫力的药物,所以我们尝试用免疫功能健全的秀丽隐杆线虫野生型N2Bristol建立液体感染模型,却由于母体被后代幼虫破坏而失败。文献提示,秀丽隐杆线虫野生型N2 Bristol可用于固体感染模型考察感染过程中白念珠菌的致病力和宿主的免疫系统的相互作用,我们成功地重现了秀丽隐杆线虫野生型N2 Bristol固体感染模型,并用白念珠菌SC5314、hog1Δ/Δ、pbs2Δ/Δ和hst7Δ/Δ进行秀丽隐杆线虫野生型N2 Bristol固体感染模型的实验检测该模型是否可以考察白念珠菌的菌丝形成能力,但是白念珠菌hog1Δ/Δ,pbs2Δ/Δ和hst7Δ/Δ感染组与SC5314感染组的生存率没有显著性的差异;用氟康唑和龙血素A进行秀丽隐杆线虫野生型N2 Bristol固体感染模型的实验检测该模型是否可以考察药物的的抗真菌作用,但是氟康唑和龙血素A药物治疗后不能提高秀丽隐杆线虫的生存率;用黄芪进行秀丽隐杆线虫野生型N2 Bristol固体感染模型的实验检测该模型是否可以考察药物的的免疫调节作用,发现黄芪药物作用后确实提高了秀丽隐杆线虫的生存率。我们推测黄芪通过提高秀丽隐杆线虫免疫力发挥抗感染的作用。即秀丽隐杆线虫野生型N2 Bristol固体感染模型不适用于考察白念珠菌的菌丝形成能力和药物的抗真菌作用,可用于考察药物的免疫调节作用。总之,龙血素A具有抗白念珠菌生物被膜的作用,秀丽隐杆线虫glp-4;sek-1液体感染可用于考察白念珠菌的致病力和药物的抗真菌作用,秀丽隐杆线虫野生型N2Bristol固体感染模型可用于考察药物的免疫调节作用。
[Abstract]:Candida albicans is found in the surface of mucosa, gastrointestinal and urogenital conditions of pathogenic fungi, when the person's immune system damage can lead to serious infection. Many infections are caused by Candida albicans in medical devices, such as stents, urethral catheterization, biofilm formation caused. The biofilm leads to repeated infections and antifungal drugs of highly resistant, so the clinical needs of anti Candida albicans biofilm drug. Antifungal drugs of animal models, but the small rat infected animal model - the common model of high cost, long experimental cycle, is not suitable for mass screening in addition, drugs, key pathogenic factors are also not suitable for batch inspection of Candida albicans. Therefore, the study of anti fungal infection requires high-throughput in vivo infection model of low cost. This paper carried out a study of the two part The first part, the effects of Loureirin A against Candida albicans biofilms, the second part of the study of Caenorhabditis elegans - Candida albicans infection model for screening anti infective agents in the pathogenicity of Candida albicans and evaluation. In the first part of the study, we use 2,3- bis (2- methoxy -4- nitro -5- -2H- four -5-) phenyl sulfonyl pyrazole formanilide method for screening anti Candida albicans biofilm drug discovery, Loureirin A has better activity. Loureirin A is isolated from the blood of the phenylpropanoid hormone drugs, dragon's blood is obtained from the resin in Dracaena. In XTT method and scanning electron microscopy method in the experiment, we found that more than 5 g/ml of Loureirin A on Candida albicans biofilm was significantly inhibited, and 5-40 g/ml of Loureirin A on Candida albicans has bactericidal and bacteriostatic effects. In more than 5 g/ml, Loureirin A can significantly reduce cell surface hydrophobicity . in more than 20 g/ml, Loureirin A inhibition in liquid and solid medium to induce the formation of mycelial medium. At the same time, the Real Time RT-PCR results showed that the adhesion of Loureirin A related genes, related genes and biological hyphae were down regulated expression of membrane associated genes. In addition, Loureirin A in G.mellonella infection model and Caenorhabditis elegans infection liquid showed anti infection effect in vivo models, and showed low toxicity. In general, Loureirin A exhibited anti Candida albicans biofilm effect, and this effect may be related to the inhibition of adhesion and hyphal formation. In the second part of the study, we investigated the beautiful elegans - Candida albicans infection in vivo model, comparison and evaluation of the solid medium is used to study the pathogenicity and drug resistance of Candida albicans infection effect of the advantages and disadvantages of Caenorhabditis elegans model based on infection of cultured in liquid Potential. First, we successfully reproduce the Caenorhabditis elegans glp-4; liquid SEK-1 infection model with Candida albicans wild-type SC5314, defective Hog1 Delta / delta mycelium, mycelium deficient pbs2 / delta and delta delta delta / hst7 deficient hyphae of Caenorhabditis elegans glp-4 infection model of SEK-1 liquid; experiments show that the model can investigate the pathogenicity of Candida albicans, Candida albicans hypase found defective Hog1 Delta / delta pbs2, Delta Delta and delta delta / hst7 / infection group, the survival rate is indeed better than the wild type SC5314 infection group; with antifungal drugs and fluconazole against Candida albicans biofilm drug - Loureirin A of Caenorhabditis elegans glp-4; SEK-1 liquid infection model experiments show that the model can evaluate drug antifungal effects of fluconazole and Loureirin A after drug treatment can improve the survival rate of Caenorhabditis elegans; immune enhancing drugs for Qi Huang Caenorhabditis elegans glp-4; experimental detection of SEK-1 infection model of the liquid immune model whether can the effects of drug regulation, but the effect of Radix Astragali drug did not improve survival in Caenorhabditis elegans. The Caenorhabditis elegans glp-4; SEK-1 fluid model can be used to test the pathogenicity of infection and drug resistance of Candida albicans by fungi the role, but the immune enhancement effect of drugs can not be investigated. Because of the Caenorhabditis elegans glp-4; SEK-1 immunodeficiency, enhance immunity drugs cannot be used for research and development, so we try to Caenorhabditis nematode of wild type N2Bristol with immune function to establish a complete liquid model for infection, but because the mother was offspring larvae damage and failure. The literature suggests that beautiful elegans wild-type N2 Bristol can be used for solid model to investigate the immune system infection infection and host pathogenicity of Candida albicans in the process of interaction, We successfully reproduce the Caenorhabditis elegans wild-type N2 solid Bristol infection model with Candida albicans SC5314, Hog1 / pbs2 / Delta Delta Delta, Delta and delta delta hst7 / tested Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can examine the hypha of Candida albicans bacteria forming ability however, Candida albicans Hog1 Delta / Delta, Delta / Delta and the difference of pbs2 hst7 Delta / delta infection group and SC5314 infection group, the survival rate was not significant; anti fungal effect detection of Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can evaluate drug use of fluconazole and Loureirin but A, fluconazole and Loureirin A after drug treatment can increase the survival rate of Xiu Liyin elegans; Huangqi tested Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can evaluate drug Immunoregulation effect of Huangqi drugs found to really improve the survival rate of Caenorhabditis elegans. We speculate that Huangqi by improving the Caenorhabditis elegans immunity play a role. The ability of anti infection and drug anti fungal effect that hyphal formation in wild-type Caenorhabditis elegans N2 Bristol solid model is not suitable for investigation of infection of Candida albicans that can be used for the immunoregulation effects of drug. In short, Loureirin A has anti Candida albicans biofilm, Caenorhabditis elegans glp-4; SEK-1 infection can be used for liquid drug to investigate the pathogenicity of Candida albicans and antifungal effects of Caenorhabditis elegans wild-type N2Bristol solid model can be used to regulate infection the immune effects of drug.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285;R-332

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