生物被膜型白念珠菌代谢物分析及代谢变化的初步研究

发布时间：2018-02-06 05:20

本文关键词： 白念珠菌海藻糖法尼醇 GC-MS HPLC-MS 代谢含量测定基因转录　出处：《第二军医大学》2010年硕士论文　论文类型：学位论文

【摘要】：代谢组学发展方兴未艾,目前代谢组学的研究已经渗透到微生物学、植物学、疾病诊断等各大邻域。本研究就是借助代谢组学的技术手段,以白念珠菌为研究对象,研究其能量代谢物的变化及初步研究其产生变化的原因。白念珠菌是一种双相型真菌,在人体中,无症状时常表现为酵母细胞型；侵犯组织和出现症状时常表现菌丝型。白念珠菌感染通常是深部感染,致死率比较高。在临床治疗过程中发现白念珠菌易形成被膜,产生耐药性,给治疗带来很大阻力。本论文运用代谢指纹分析方法,以GC-MS为分析手段研究白念珠菌的代谢物,区别浮游型和被膜型白念珠菌,并通过化学计量学方法对图谱进行分析,推出两者可能的差异代谢物。法尼醇(Farnesol)是抑制白念珠菌由酵母型向菌丝型转变的一种感应分子,它由白念珠菌产生并排到细胞外,研究表明在培养基中外加感应分子法尼醇可以抑制白念珠菌生物被膜的形成。海藻糖(Trehalose)是细胞中的一种应激代谢物,在细胞处于应激时可大量合成,恢复平常状态下又迅速降解。海藻糖含量的变化从某种程度上反映了机体对外界环境的响应。由于糖的组分复杂,结构相似,加之糖没有显色基团,难以不经衍生就进行光谱、色谱分析,因此,糖的分析难度较大。本研究正是建立了一种简便快速准确的测定海藻糖的方法,用来测定法尼醇干扰下被膜型白念珠菌中海藻糖的含量变化,结果发现法尼醇的干预确实会引起海藻糖的含量波动。为了解释海藻糖含量波动的原因,又从白念珠菌细胞中海藻糖的代谢循环着手研究。虽然其代谢循环过程比较简单,但却是由复杂的调控机制所调控。本文研究调控代谢过程的酶的基因——hxk、tps1、tps2、ath1、nth、zwf,研究它们在法尼醇作用下基因的转录水平的变化。研究表明,这六种基因转录水平差异明显,部分解释了海藻糖含量变化的原因。本课题建立了海藻糖的含量测定方法,研究了白念珠菌被膜形成过程中细胞内法尼醇含量的变化以及外加法尼醇对细胞内海藻糖生成的影响,为白念珠菌耐药性研究提供了参考。
[Abstract]:The development of metabonomics is in the ascendant. At present, the research of metabonomics has penetrated into the microbiology, botany, disease diagnosis and so on. This study is based on the technical means of metabonomics. The changes of energy metabolites of Candida albicans and their causes were studied. Candida albicans is a biphasic fungus. The infection of Candida albicans is usually deep infection with high mortality. In the course of clinical treatment, Candida albicans are found to be prone to the formation of capsule and drug resistance. Bring a lot of resistance to the treatment. In this paper, the metabolites of Candida albicans were studied by GC-MS, and the phytoplankton and membranous Candida albicans were distinguished, and the chromatogram was analyzed by chemometrics. A possible differential metabolite between the two is suggested. Farnesol is an inductive molecule that inhibits the transformation of Candida albicans from yeast to hyphae. It is produced by Candida albicans and discharged out of cells. The results showed that the addition of faniol could inhibit the formation of biofilm in Candida albicans. Trehalose (Trehalose) is a stress metabolite in cells, which can be synthesized in large quantities when the cells are under stress. The changes of trehalose content reflect the response of the body to the external environment to some extent. The sugar composition is complex, the structure is similar, and the sugar has no chromogenic group. It is difficult to carry out spectrum and chromatographic analysis without derivation, so the analysis of sugar is difficult. This study is to establish a simple, rapid and accurate method for the determination of trehalose. The changes of trehalose content in membranous Candida albicans under the interference of mannitol were determined. The results showed that the intervention of mannitol did cause the fluctuation of trehalose content. In order to explain the fluctuation of trehalose content, the metabolic cycle of trehalose in Candida albicans was studied. However, it is regulated by complex regulatory mechanisms. In this paper, we studied the gene of the enzyme regulating metabolic process, hxktps1, tps2ath1, nththzwf. The results showed that the transcriptional levels of these six genes were significantly different, which partly explained the change of trehalose content. In this paper, a method for the determination of trehalose was established, and the changes of intracellular mannitol content during the formation of Candida albicans membrane and the effect of addition of farnesol on intracellular trehalose production were studied. It provides a reference for the study of resistance of Candida albicans.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【引证文献】