白念珠菌酵母相和菌丝相细胞表达基因谱的构建及聚类分析

发布时间：2018-03-20 05:05

本文选题：白念珠菌　切入点：二态性　出处：《第三军医大学》2005年博士论文　论文类型：学位论文

【摘要】：近年来念珠菌感染已成为住院患者血行感染中第三或第四位最常见的病原菌,其中白念珠菌最为常见,占念珠菌属40-70%。白念珠菌属人体常居真菌,为条件致病真菌,一般以酶母相存在于机体,当机体免疫功能受到抑制或微生态失衡时,白念珠菌会转变其生长形态,可由酵母相转为菌丝相,进而对机体产生危害。体外培养条件下,白念珠菌也可以酶母相形式或菌丝相形式生长,这种酵母与菌丝之间的转换称为菌相转换。正因为白念珠菌在医学中的重要地位,因此对其致病的机制进行了广泛深入的研究。近年来对白念珠菌的毒力因子研究主要集中于三个方面:①粘附;②酶类;③菌相转换。菌相转换,即酵母相生长转到菌丝相生长的这一变化是白念珠菌感染转变的过程,菌丝多则感染率高;另一方面,当天然突变或人工中断某些基因导致不能形成菌丝的白念珠菌突变菌株,则在动物实验中表现出毒力的降低或丧失,因此白念珠菌菌丝的形成与发病有一定的关系。已有的研究证实,菌丝的形成改变白念珠菌各种毒力因子的表达,致菌株毒力增加,感染性增强。为更全面的寻找白念珠菌酵母相和菌丝相致病相关基因及其毒力因子,本研究应用LongSAGE 技术,定性定量检测白念珠菌酵母相和菌丝相细胞表达基因的改变,构建白念珠菌酵母相和菌丝相细胞表达基因谱,聚类分析表达基因功能,探讨酵母相和菌丝相细胞表达基因与菌相转换、菌株毒力的相关性。本研究选择白念珠菌ATCC-90028 作为研究菌株,经RPMI1640 体外诱导形成纯的酵母相和菌丝相细胞作为研究材料,经显微镜下计数其纯度,细胞纯度超过95%用于提取RNA;提取纯化细胞的RNA 经RT-PCR 检测,证实实验菌株为白念珠菌,且酵母相细胞无菌丝相细胞污染后,使用分离的RNA 进行LongSAGE 标签库的构建,这种使用细胞表型分子与特异性表达基因的双重筛选,确保RNA 来源于均一性细胞,使经LongSAGE 标签库分析获得的基因表达谱,能够代表该细胞的基因表达特征。
[Abstract]:In recent years, Candida albicans infection has become the third or 4th most common pathogen in blood infection of hospitalized patients, among which Candida albicans is the most common, accounting for 40-70th of Candida. When the immune function of the body is inhibited or the microecology is out of balance, Candida albicans will change its growth form, which can be changed from yeast to hyphae, and then harm to the body. Candida albicans can also grow in the form of an enzyme or a hyphae, and this transition between yeast and hyphae is called phase transition, precisely because of the importance of Candida albicans in medicine. In recent years, the virulence factors of Candida albicans were mainly focused on three aspects: 1: 1 adhesion to 2 enzymes and 3 strains. The transformation of mycelia from yeast to hyphae is the process of infection transformation of Candida albicans, and the infection rate of mycelium is high. When a mutant strain of Candida albicans that is unable to form hyphae due to natural mutation or artificial interruption of certain genes, it shows a decrease or loss of virulence in animal experiments. Therefore, the formation of Candida albicans mycelium has a certain relationship with the development of mycelium, which has been confirmed that the formation of mycelium changes the expression of various virulence factors of Candida albicans, resulting in increased virulence and increased infectivity of Candida albicans. In order to search for the pathogenicity genes and virulence factors of Candida albicans yeast and hyphae more comprehensively, the expression genes of yeast and hyphae of Candida albicans were detected qualitatively and quantitatively by LongSAGE technique. The cell expression gene profiles of Candida albicans in yeast and hyphal phase were constructed, and the function of expressed genes was analyzed by cluster analysis. The relationship between the expression of genes in yeast and hyphae cells and the transformation between yeast and mycelium and the virulence of the strain was discussed. In this study, Candida albicans ATCC-90028 was selected as the study strain, and pure yeast and hyphal phase cells were induced by RPMI1640 in vitro, and their purity was counted under microscope. The RNA of purified cells was identified as Candida albicans by RT-PCR detection, and the isolated RNA was used to construct the LongSAGE tagging library after the yeast phase cells were contaminated with hyphae cells. The double screening of cell phenotypic molecules and specific expressed genes ensures that RNA is derived from homogeneous cells, so that the gene expression profiles obtained by LongSAGE tagging library can represent the gene expression characteristics of the cells.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R379

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