白念珠菌HOG基因致病相关性研究

发布时间：2018-02-01 07:07

本文关键词： 白念珠菌 HOG基因超微结构药敏实验小鼠模型毒力　出处：《福建医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的: 比较白念珠菌HOG21(hog/hog双等位基因缺失株)、CHK21(chk/chk双等位基因缺失株)和标准株CAF-2菌株细胞壁超微结构、对氟康唑MIC值及在系统性感染小鼠模型中致病力的差异,探讨HOG基因在白念珠菌致病机制中的作用及其与CHK基因的关系,旨在寻找系统性白念珠菌感染致病相关基因,筛选新的抗真菌药物作用靶点,为今后研发出更高效、低毒、广谱的新型抗真菌药物提供理论依据。方法: 1.建立系统性白念珠菌感染小鼠模型,比较死亡率并描绘生存曲线;平皿稀释法检测各组脾肾组织菌落形成单位(CFU)数目;制作肾组织病理学标本,评估真菌感染程度。 2.利用扫描和透射电镜观察三株菌株细胞壁超微结构的差异。 3.微量稀释法检测三株菌株对氟康唑MIC值的差异。结果: 1.体内实验: (1)成功建立系统性白念珠菌感染小鼠模型。 (2)CAF-2组、CHK21组和HOG21组感染小鼠观察期间死亡率分别为100%、0、0,差异有统计学意义。 (3)脾肾组织菌落形成单位(CFU)结果:各观察时间点CAF-2组脾肾组织带菌量(CFU)比HOG21组和CHK21组高,差异有统计学意义;HOG21组与CHK21组比较差异无统计学意义。 (4)组织病理学观察结果:CAF-2组肾髓质区见弥漫性菌丝和孢子,伴人多形核粒细胞(PMN)为主的炎性细胞浸润,肾组织结构破坏严重;HOG21组肾乳头见少量菌丝和孢子被PMN包裹形成局限性脓肿;CHK21组则表现为以肾脏髓腔内PMN浸润为主。电镜结果:CAF-2组肾小管上皮细胞微绒毛破坏严重;HOG21组肾小管上皮细胞内见被吞噬的孢子;而CHK21组肾组织病变较轻。 2.体外实验: (1)扫描电镜下,CAF-2表面光滑;CHK21表面见小斑块样突起;HOG21表面见细小点状突起。透射电镜下,CAF-2胞壁结构完整,电子致密层、电子透明层及细胞膜各层结构清晰可见;CHK21细胞壁可见电子致密层变薄甚至局灶性缺失;HOG21细胞壁电子透明层厚薄不一,部分见电子致密层局灶性缺失、细胞膜外突、不连续以及胞膜周围囊泡聚集的现象。 (2)微量稀释法检测各组酵母细胞对氟康唑MIC值:CAF-2:1ug/ml;CHK21:0.125~0.25ug/ml;HOG21:0.25ug/ml。结论: 1. HOG基因参与白念珠菌致病性的调控过程。 2. HOG基因参与调控白念珠菌细胞壁的生物合成。 3. HOG基因影响白念珠菌对氟康唑的敏感性,这可能为新型药靶的筛选和或新型药靶抑制剂与氟康唑联合运用进行高效、低毒抗真菌治疗提供理论依据。 4.CHK基因也参与对白念珠菌毒力、细胞壁的生物合成以及对氟康唑敏感性的调控过程,但其作用机制可能有别于HOG基因。
[Abstract]:Objective: To compare the two allelic deletion strains of Candida albicans HOG21(hog/hog). The ultrastructure of cell wall, MIC value of fluconazole and pathogenicity of CAF-2 strain in systemic infection mice. To explore the role of HOG gene in the pathogenesis of Candida albicans and its relationship with CHK gene, the aim of this study was to search for the pathogenic genes associated with systemic Candida albicans infection and to screen new antifungal drug targets. It provides theoretical basis for developing new antifungal drugs with high efficiency, low toxicity and broad spectrum. Methods: 1. Establish systemic Candida albicans infected mice model, compare mortality and depict survival curve; The number of colony forming units (CFU) of spleen and kidney tissue in each group was detected by plate dilution method. Renal histopathological specimens were made to evaluate the degree of fungal infection. 2. The ultrastructure of cell wall of three strains were observed by scanning and transmission electron microscopy. 3.The difference of fluconazole MIC between three strains was detected by microdilution method. Results: 1. In vivo experiments: 1) the model of systemic Candida albicans infection in mice was established successfully. The mortality of infected mice in CAF-2 group and CHK21 group and HOG21 group were 100 and 0, respectively, and the difference was statistically significant. (3) CFU (colony forming unit of spleen and kidney): the amount of CFU in spleen and kidney tissue of CAF-2 group was higher than that of HOG21 group and CHK21 group at different observation time points. The difference was statistically significant. There was no significant difference between HOG21 group and CHK21 group. The histopathological results showed that diffuse hyphae and spores were found in medulla of the kidney in group 1: CAF-2, accompanied by infiltration of inflammatory cells dominated by human polymorphonuclear granulocyte (PMN), and serious damage of renal tissue structure. In HOG21 group, a small number of hyphae and spores were encapsulated by PMN to form localized abscess. In CHK21 group, intramedullary PMN infiltration was dominant. The microvilli of renal tubular epithelial cells were severely damaged in the CHK21 group. In HOG21 group, phagocytic spores were found in renal tubular epithelial cells. In CHK21 group, renal tissue lesion was mild. 2. In vitro experiments: 1) the surface of CAF-2 was smooth under scanning electron microscope. Small plaques were seen on the surface of CHK21. The surface of HOG21 showed small punctate protrusions. The wall structure of CAF-2 was intact, the electron dense layer, the electron transparent layer and each layer of cell membrane were clearly visible under transmission electron microscope. In the CHK21 cell wall, the electron dense layer was thinned and even the focal absence was observed. The thickness of the electron transparent layer of HOG21 cell wall was different, and some of the electron dense layers were found to have focal absence, extracellular protrusion, discontinuity and the accumulation of vesicles around the cell membrane. (2) Microdilution method was used to detect the MIC value of fluconazole cells in each group: CAF-2: 1ug-mml. CHK 21: 0.125 U / ml; HOG21:0.25ug/ml. Conclusion: 1. HOG gene is involved in the regulation of the pathogenicity of Candida albicans. 2. HOG gene is involved in the regulation of cell wall biosynthesis of Candida albicans. 3. HOG gene affects the sensitivity of Candida albicans to fluconazole, which may be a high efficiency for the screening of new drug targets and the combination of new target inhibitors and fluconazole. Low toxicity antifungal therapy provides theoretical basis. 4. CHK gene is also involved in the virulence of Candida albicans, biosynthesis of cell wall and regulation of fluconazole sensitivity, but its mechanism may be different from that of HOG gene.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379.4

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