白念珠菌新基因IPF19998的氧化还原相关功能研究

发布时间：2018-01-21 11:16

本文关键词： 白念珠菌线粒体基因敲除生物被膜内源性活性氧　出处：《第二军医大学》2010年硕士论文　论文类型：学位论文

【摘要】：白念珠菌易于感染、难以防治的特点使其在临床治疗中表现为高致病性和高耐药性。目前国际上对白念珠菌基因组的测序工作已经完成,并可以通过公共数据库可以获得相应的完整基因组序列。经过DNA序列及编码的氨基酸序列比对,发现一个白念珠菌新基因IPF19998与酿酒酵母中的AIF1基因有高度同源性。AIF1是与人类AIF同源的酵母细胞凋亡诱导因子(Apoptosis inducing factor)。已经证实在酿酒酵母中,在由H2O2或醋酸盐诱发的凋亡以及衰老过程中,Aif1p从线粒体中转位至细胞核内,并诱导细胞凋亡。在AIF1基因敲除菌株中,凋亡特征消失,衰老延缓[1]。由此我们推测,IPF19998是白念珠菌中的凋亡诱导因子,并在白念珠菌细胞凋亡过程中发挥重要作用,对该基因的进一步研究有助于阐明白念珠菌细胞凋亡机制,为抗真菌感染药物的研究设计提供新思路。本课题采用Ura-Blaster基因敲除策略靶向敲除和pCaEXP质粒异位表达凋亡诱导因子基因IPF19998,构建了IPF19998基因缺失菌和高表达菌。将构建好的菌株通过比浊法测定生长曲线;通过微量液基实验来考察IPF19998缺失菌对唑类药物的敏感性及过氧化氢的刺激耐受性是否改变;将缺失菌在塑料培养板中按照生物被膜形成条件培养24小时并形成生物被膜,考察其生长动力学变化;通过荧光染料检测IPF19998基因缺失菌在酵母态及被膜态两种状态下的内源性活性氧水平(ROS),通过实时定量PCR考察了IPF19998基因缺失后,其他已知氧化还原相关基因和生物被膜形成相关基因表达量的变化。结论:成功构建白念珠菌IPF19998基因缺失菌和基因高表达菌株;IPF19998基因缺失后,菌株生长速度不变;但对唑类药物敏感性没有明显变化,高表达菌株对唑类药物产生耐药性;对过氧化氢刺激所引起的凋亡也与野生菌相比,无明显差异。这与我们前期设想的结论不同。但用荧光染料测其内源性活性氧水平(ROS),与酵母态下生成的ROS含量相比,被膜态下的菌株,不论是敲除菌株,高表达菌株还是野生型菌株,其ROS产生远远低于酵母态下的ROS产生。而在被膜态下,IPF19998基因缺失菌ROS产生增多,而高表达菌ROS产生保持一个稳定且低的水平。同时,我们发现,IPF19998基因缺失以后,其形成生物被膜的能力下降,高表达菌株生成生物被膜的能力明显增强。这一结果在Real-time实验中也得到证实。IPF19998基因能改变已知氧化还原相关基因和生物被膜形成相关基因表达量的变化。根据本研究推测,IPF19998基因可通过增强抗氧化能力,清除细胞内的ROS含量,增强生物被膜的形成能力,从而降低对药物的敏感性。与预测的促进细胞凋亡功能无关,而与生物被膜形成过程中的抗氧化能力有关。具体机制有待进一步探讨。
[Abstract]:Candida albicans are susceptible to infection and difficult to prevent and cure, which makes them show high pathogenicity and high drug resistance in clinical treatment. At present, the genome sequencing of Candida albicans has been completed. The complete genome sequence, DNA sequence and amino acid sequence alignment can be obtained by common database. A novel Candida albicans gene IPF19998 has high homology with AIF1 gene in Saccharomyces cerevisiae. AIF1 is a yeast apoptosis inducing factor homologous to human AIF. Apoptosis inducing factor.has been confirmed in Saccharomyces cerevisiae. Apoptosis induced by H2O2 or acetate and translocation of Aif1p from mitochondria to nucleus during senescence induced apoptosis. In AIF1 gene knockout strains, the apoptotic characteristics disappeared. Aging delay. [1. We speculate that IPF19998 is an apoptosis-inducing factor in Candida albicans and plays an important role in the apoptosis process of Candida albicans. The further study of this gene is helpful to elucidate the apoptosis mechanism of Candida albicans and provide a new idea for the research and design of antifungal agents. In this study, Ura-Blaster gene knockout strategy and pCaEXP plasmid heterotopic expression of apoptosis-inducing factor gene IPF19998 were used. IPF19998 gene deficient bacteria and high expression bacteria were constructed. The growth curves of the constructed strains were determined by turbidimetry. The sensitivity of IPF19998 deficient bacteria to azolides and the tolerance to hydrogen peroxide stimulation were investigated by microamount liquid based test. The absence bacteria were cultured in plastic culture plate for 24 hours according to the conditions of biofilm formation, and the biofilm was formed, and the growth kinetics was investigated. The endogenous reactive oxygen species (Ros) of IPF19998 gene deficient bacteria in yeast and membranous state were detected by fluorescent dye. The deletion of IPF19998 gene was investigated by real-time quantitative PCR. Changes in the expression of other known redox related genes and biofilm formation genes. Conclusion: Candida albicans IPF19998 gene deletion and gene overexpression strains were successfully constructed. After the deletion of IPF19998 gene, the growth rate of the strain was unchanged. However, there was no significant change in the sensitivity to the zolium drugs, and the high expression strains developed drug resistance to the zolium drugs. The apoptosis induced by hydrogen peroxide was not significantly different from that of wild bacteria, which was different from the previous conclusion, but the endogenous reactive oxygen species level was measured with fluorescent dyes. Compared with the ROS content produced by yeast, the strains under membrane condition, whether knockout, high expression or wild-type strains. The production of ROS was much lower than that of ROS in yeast, but the ROS production of IPF19998 gene deletion strain increased in the membranous state. Moreover, we found that the ability to form biofilm was decreased after the deletion of ROS. The ability of highly expressed strain to produce biofilm was significantly enhanced. This result was also confirmed in Real-time experiments. IPF19998 gene can change known redox related genes and biofilm. The changes in the expression of related genes. IPF19998 gene can enhance the ability of biofilm formation by enhancing the antioxidant ability, clearing the content of ROS in the cells and enhancing the ability of biofilm formation. It is not related to the predicted function of promoting apoptosis, but related to the antioxidant ability during the formation of biofilm. The specific mechanism needs to be further explored.
【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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