饥饿状态对白色念珠菌生物膜滞留菌的影响及滞留菌相关基因的单细胞分析

发布时间：2018-03-28 02:03

本文选题：饥饿状态　切入点：白色念珠菌生物膜　出处：《山东大学》2014年硕士论文

【摘要】：目的作为人类最常见的条件致病菌——白色念珠菌(Candida albicans),所引起的感染性疾病大都是慢性和难治性的,这与其形成的生物膜结构密切相关。白色念珠菌生物膜高耐药性的可能机制有：生物膜基质对药物的空间位阻和屏障作用；生物膜中白色念珠菌的特殊生长状态,例如迟缓生长状态；基因水平的耐药机制；生物被膜内细胞质膜脂质成分的影响；以及近几年提出的‘滞留菌’的产生等等。越来越多的研究提示,正是滞留菌(Persisters)的存在,使得药物无法完全清除白色念珠菌生物膜,对慢性迁延性感染的产生起到了关键性的作用。已有研究证实了细菌滞留菌的产生机制主要包括以下2个方面：微生物群体中生长速度的异质性,极少数生长处于静止状态的菌细胞亚群成为滞留菌；饥饿等环境因素导致的SOS反应,即极端不利环境因素诱导。这表明滞留菌的产生机制,并非单一的由生长状态异质性造成的,而是与环境因素的诱导也有密切关系。那么,改变生物膜代谢的营养环境,是不是也会对白色念珠菌耐药滞留菌的产生有一定的影响呢? 本实验即通过研究饥饿环境对白色念珠菌生物膜以及其耐药滞留菌形成的影响,证实白色念珠菌生物膜耐药滞留菌形成的第二种机制(环境因素影响机制)；并且,进一步运用基于Taqman探针的单细胞real-time RT-PCR技术相对定量检测滞留菌相关基因,为今后白色念珠菌耐药滞留菌相关基因的研究提供方便、可行的研究技术。材料和方法 (1)饥饿状态对白色念珠菌生物膜及其耐药滞留菌形成的影响体外分别构建对数期、稳定期和饥饿状态白色念珠菌生物膜模型,两性霉素B(AmB)冲击作用24h,冲击前后均进行活菌菌落计数(CFU),计算得出滞留菌比例并进行统计学分析,同时通过共聚焦显微镜观察和分析各组生物膜的形成状况。 (2)基于Taqman探针的单细胞real-time RT-PCR技术相对定量检测滞留菌相关基因的表达水平倒置显微镜下,使用自行拉制的分离微吸管吸取非滞留菌细胞和滞留菌细胞,进行基于Taqman探针的单细胞real-time RT-PCR,相对定量检测白色念珠菌生物膜耐药滞留菌EFG1、ERG2、MKC1、GIN4基因的表达水平,采用SPSS11.70软件包对数据进行统计学分析。结果 (1)对数期、稳定期和饥饿状态白色念珠菌生物膜的形成能力依次下降(P0.05),具有显著性差异,对数期和稳定期白色念珠菌生物膜形成的为菌丝相生物膜,而饥饿态白色念珠菌形成的为酵母相生物膜。药物作用后滞留菌比例依次上升(P0.05)。 (2)基于Taqman探针的单细胞real-time RT-PCR技术相对定量地检测出了白色念珠菌滞留菌中EFG1、ERG2、MKC1、GIN4的表达水平。其中,除了MKC1在滞留菌细胞和非滞留菌细胞中的表达水平无显著性差异(P0.05)外,EFG1、 ERG2、GIN4在滞留菌中的表达水平明显升高(P0.05),具有显著性差异。结论 (1)营养状态对白色念珠菌滞留菌的产生有影响,饥饿状态能够提高滞留菌的产生比例,提示白色念珠菌耐药滞留菌形成与营养状态等环境因素有关。 (2)自制的分离微吸管能够成功分离出滞留菌细胞,在此基础上,基于Taqman探针的单细胞real-time RT-PCR技术是快速检测滞留菌相关基因表达水平的有效方法,为今后研究白色念珠菌滞留菌相关基因提供了方便、可行的研究技术。 (3)白色念珠菌滞留菌的形成与基因EFG1、ERG2以及GIN4均密切相关,并非由单一的信号通路决定的,受多条信号途径的调节。
[Abstract]:objective
As one of the most common human opportunistic pathogen, Candida albicans (Candida albicans), infectious diseases caused by mostly chronic and refractory, closely related with the formation of the biofilm structure. The possible mechanism of Candida albicans biofilm high resistance: space biofilm matrix of drug resistance and barrier special effects; growth state in biofilms of Candida albicans, such as slow growth state; mechanism of resistance gene level; biological effect of cytoplasmic membrane lipid composition in the film; and put forward in recent years' persisters' production and so on. More and more studies suggest that it is the presence of persisters (Persisters) the drug, unable to completely remove the Candida albicans biofilm in chronic persistent infection have played a key role
Use.
Studies have confirmed the mechanism of bacterial retention of bacteria mainly include the following 2 aspects: the heterogeneity of the growth rate of the microbial population growth, a handful of quiescent cell subsets become stranded bacteria; SOS reaction led to starvation and other environmental factors, in which the extreme adverse environmental factors. This indicates that the mechanism of induced retention of bacteria that is not a single state of growth caused by heterogeneity, induced and environmental factors but also have close relationship. So, nutrition environment change membrane metabolism, is not also on Candida albicans resistance retention producing bacteria have certain effect?
Effect of this experiment through the study of starvation on Candida albicans biofilm and the resistance of persisters formation, second mechanisms that Candida albicans biofilm formation (resistant bacteria retention effect mechanism of environmental factors); and further use of the relative quantitative detection of single cell real-time RT-PCR Taqman probe persisters related genes based on convenience for the future study of Candida albicans resistant bacteria retention related genes, the research technique is feasible.
Materials and methods
(1) the effect of starvation on the formation of Candida albicans biofilm and its drug-resistant stranded bacteria
In vitro constructed logarithmic phase, stable phase and starvation of Candida albicans biofilm model, amphotericin B (AmB) 24h impact, the impact of both before and after the live bacteria colony count (CFU), calculated the proportion of persisters were statistically analyzed and observed by confocal microscopy and analysis of each biofilm formation.
(2) single cell real-time RT-PCR technique based on Taqman probe to quantitatively detect the expression level of retained bacteria related genes
Under the microscope, using separate self drawn micro Straw from non persister cells and retention of bacterial cells, Taqman probe for single cell real-time RT-PCR based on the relative quantitative detection of Candida albicans biofilm drug resistance bacteria EFG1 ERG2, MKC1 retention, and the expression level of GIN4 gene, package the data were analyzed using SPSS11.70 software.
Result
(1) the logarithmic phase, stable phase and starvation formation of Candida albicans biofilm decreased (P0.05), with a significant difference, log phase and stable phase of Candida albicans biofilm formation for hyphal biofilm, and the hunger for the formation of Candida albicans biofilm. The yeast phase after the drug retention of bacteria in order to rise the proportion (P0.05).
(2) single cell real-time RT-PCR technology of Taqman probe quantitative to detect Candida albicans bacteria retention based on ERG2, EFG1, MKC1, the expression level of GIN4. Among them, in addition to MKC1 was significantly higher in the persister cells and non expression level differences in bacterial cell retention (P0.05), EFG1, ERG2. The expression level of GIN4 in residence in bacteria increased significantly (P0.05), with a significant difference.
conclusion
(1) nutritional status has an effect on the production of Candida albicans, and starvation can increase the proportion of the remaining bacteria, indicating that the occurrence of drug-resistant bacteria in Candida albicans is related to environmental factors such as nutritional status.
(2) the self-made micro Straw separation can be successfully isolated stranded bacterial cells, on this basis, based on the single cell real-time RT-PCR technology of Taqman probe is rapid detection of persisters related gene expression level of effective methods, for the future study of Candida albicans persisters related gene research provides a convenient, feasible technology.
(3) the formation of Candida albicans is closely related to the genes EFG1, ERG2 and GIN4. It is not determined by a single signaling pathway, but regulated by multiple signaling pathways.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.54

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