白念珠菌MIR1基因的功能研究

发布时间：2018-02-12 00:46

本文关键词： 白念珠菌耐药性 MIR1 基因敲除　出处：《第二军医大学》2010年硕士论文　论文类型：学位论文

【摘要】：白念珠菌的耐药性产生已成为临床白念珠菌感染治疗失败的主要原因。本课题组长期致力于白念珠菌耐药机制的研究,我们前期的研究结果提示MIR1和白念珠菌耐药性的形成密切相关。本课题利用基因敲除技术系统性的考察了MIR1的功能：采用Ura-Blaster策略将MIR1的两个等位基因从白念珠菌的基因组中敲除,构建了MIR1基因缺失菌继而用以研究MIR1的功能。研究结果显示：MIR1基因敲除后白念珠菌生长速度减慢；通过生物被膜的生长动力学测定(XTT法)、激光共聚焦显微镜(CSLM)观察生物被膜和细胞表面疏水性(CSH)测定等方法考察了MIR1对白念珠菌菌丝和生物被膜形成能力的影响,结果显示MIR1基因缺失菌形成菌丝和被膜的能力下降,MIR1基因减弱生物被膜和菌丝的生成能力。上述结果提示：MIR1基因在白念珠菌的生物被膜形成中发挥重要作用。考察MIR1基因对药物敏感性的影响：实验结果显示MIR1基因对氟康唑、酮康唑和特比奈芬药物敏感性降低,Spot assay和Filter disk assay结果一致。随后我们对基因敲除后药物敏感性下降的机制进行了探讨。罗丹明6G实验结果显示：MIR1敲除后白念珠菌对药物外排能力增加,即药物外排泵的功能增强；但是检测一系列耐药相关基因结果发现,耐药相关基因的转录表达没有发生明显变化。因为罗丹明6G是能量依赖型物质外排,所以考察了细胞内ATP的水平,结果发现：MIR1基因敲除后,细胞内ATP水平升高；一般情况下,有氧呼吸的线粒体呼吸链是细胞内ATP的主要来源,考察线粒体膜电位水平发现：MIR1基因敲除后,线粒体功能的直接指标——线粒体膜电位升高。内源性活性氧(ROS)超过一定浓度会对自身产生氧化损伤,是唑类药物的主要抑菌因素之一；同时线粒体呼吸链是细胞内源性活性氧的主要来源,对MIR1基因敲除后进行内源性活性氧测定,结果发现：MIR1敲除后,白念珠菌内源性活性氧的增加程度低于亲本菌。综上所述,MIR1基因敲除后白念珠菌对药物敏感性降低的原因,一方面是由于药物外排泵的能量增加,使其功能增强；另一方面原因是敲除菌的内源性活性氧的增加程度低于亲本菌。本课题从多方面、多层次阐明,MIR1影响白念珠菌诱导耐药性的产生。
[Abstract]:The drug resistance of Candida albicans has become the main reason for the failure of clinical treatment of Candida albicans infection. Our previous results suggest that MIR1 is closely related to the formation of resistance to Candida albicans. This study systematically investigated the function of MIR1 by using gene knockout technique: Ura-Blaster strategy was used to remove two alleles of MIR1 from Candida albicans. Knockout in the genome, MIR1 gene deletion bacteria were constructed and used to study the function of MIR1. The results showed that the growth rate of Candida albicans was slowed down after the knockout of the 1: MIR1 gene. The effects of MIR1 on the formation of mycelium and biofilm of Candida albicans were investigated by means of the growth kinetics of biofilm and the observation of cell surface hydrophobicity by laser confocal microscope (CSLM) and the growth kinetics of biofilm. The results showed that the ability of MIR1 gene deletion bacteria to form mycelium and membrane decreased. The results indicated that the cell membrane formation of Candida albicans was played an important role in the formation of biofilm of Candida albicans by the reduction of biofilm and mycelium formation by MIR1 gene. The effect of MIR1 gene on drug sensitivity: the results showed that MIR1 gene had effect on fluconazole, The results of ketoconazole and terbinafine decreased drug sensitivity were consistent with those of Filter disk assay. Then we discussed the mechanism of drug sensitivity decrease after gene knockout. Rhodamine 6G experiment showed that Candida albicans after 1% MIR1 knockout. Increased drug efflux capacity, In other words, the function of drug efflux pump was enhanced, but the transcriptional expression of drug-resistance related genes was not changed significantly by detecting a series of drug-resistance related genes, because Rhodamine 6G was an energy-dependent efflux. So when we examined the level of ATP in cells, we found that the level of ATP increased after the knockout of the 1: MIR1 gene. In general, the mitochondrial respiratory chain of aerobic respiration was the main source of intracellular ATP. It was found that the mitochondrial membrane potential, the direct index of mitochondrial function, increased after the 1: MIR1 gene knockout. More than a certain concentration of endogenous reactive oxygen species (Ros) caused oxidative damage to the mitochondria. The mitochondrial respiratory chain was the main source of endogenous reactive oxygen species (Ros) in cells. The endogenous reactive oxygen species (Ros) were determined after knockout of the MIR1 gene, and the results showed that after 1% MIR1 knockout, the mitochondrial respiratory chain was the main source of endogenous reactive oxygen species (Ros). The increase of endogenous reactive oxygen species in Candida albicans was lower than that of parent strains. In conclusion, the reason for the decrease of drug sensitivity of Candida albicans after gene knockout of MIR1 gene was that, on the one hand, the energy of drug efflux pump increased and the function of Candida albicans increased. On the other hand, the increase of endogenous reactive oxygen species in knockout bacteria is lower than that of parent bacteria.
【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】：R379

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