【摘要】： 白念珠菌疾病易于感染、難以防治的特點(diǎn)使其在臨床治療中表現(xiàn)為強(qiáng)烈的致病性和高耐藥性。前期研究顯示白念珠菌耐藥性形成過程中可能伴隨著線粒體氧化呼吸功能的改變,并發(fā)現(xiàn)與線粒體呼吸相關(guān)的基因MRF1在耐藥株中表達(dá)上調(diào)。為了確證白念珠菌耐藥形成的“呼吸體抑制”假說,進(jìn)一步闡明白念珠菌的耐藥形成機(jī)制。本課題采用Ura-Blaster基因敲除策略靶向敲除和pCaEXP質(zhì)粒異位表達(dá)線粒體呼吸功能相關(guān)的基因MRF1,構(gòu)建了MRF1基因缺失菌和高表達(dá)菌。將構(gòu)建好的菌株通過比濁法測定生長曲線；通過spot assay和微量液基實(shí)驗(yàn)來考察MRF1缺失菌對唑類藥物的敏感性及過氧化氫的刺激耐受性是否改變；通過熒光染料檢測MRF1基因缺失菌的線粒體膜電位、ATP產(chǎn)生和內(nèi)源性活性氧水平(ROS),通過實(shí)時(shí)定量PCR考察了MRF1基因高表達(dá)后,其他已知耐藥相關(guān)基因表達(dá)量的變化；又考察了MRF1缺失菌生物被膜、菌絲的形成能力、細(xì)胞膜甾醇成份和外排能力的改變情況,較全面的研究了MRF1基因的相關(guān)功能。結(jié)論：成功構(gòu)建白念珠菌MRF1基因缺失菌和基因高表達(dá)菌；MRF1基因缺失后：菌株線粒體膜電位、ATP含量略降低,菌株生長速度延緩、對氧化刺激敏感性略增加、ROS產(chǎn)生略增多。釀酒酵母中MRF1編碼的Ybr026p為2-烯酰硫酯還原酶,它催化線粒體脂肪酸合成通路Ⅱ的最后一步反應(yīng),產(chǎn)生作為線粒體膜成份之一的脂肪酸及合成硫辛酸的前體。由于白念MRF1p與Ybr026p同源,因此我們推測上述結(jié)果是因?yàn)镸RF1缺失后不能正常合成2-烯酰硫酯還原酶,造成線粒體膜一定程度上的破損,細(xì)胞色素內(nèi)容物降低,進(jìn)而減弱呼吸作用而導(dǎo)致的。但我們通過測定基因缺失菌的藥物MIC80值和spot assay實(shí)驗(yàn),發(fā)現(xiàn)其對唑類藥物的耐藥性并沒有顯著性的改變,也不影響細(xì)胞膜的甾醇含量和外排能力,這一現(xiàn)象在Real-time結(jié)果中也得以證實(shí)。由于線粒體膜的功能維持涉及了大量的編碼因子,推測MRF1基因非其決定性因素,所以其缺失不能對白念線粒體呼吸產(chǎn)生明顯的抑制作用。此外,本研究發(fā)現(xiàn)白念珠菌MRF1基因能明顯抑制細(xì)胞菌絲和被膜的形成,激光共聚焦實(shí)驗(yàn)也驗(yàn)證了這一現(xiàn)象,具體機(jī)制有待進(jìn)一步探討。
[Abstract]:Candida albicans is susceptible to infection and difficult to prevent and cure, which makes it strong pathogenicity and high drug resistance in clinical treatment. Previous studies have shown that the formation of resistance of Candida albicans may be accompanied by changes in mitochondrial oxidative respiratory function, and the expression of mitochondrial respiratory related gene MRF1 is up-regulated in drug-resistant strains. In order to confirm the hypothesis of "respiratory inhibition" caused by Candida albicans resistance, the mechanism of drug resistance of Candida albicans was further elucidated. In this study, MRF1 gene deletion bacteria and high expression bacteria were constructed using Ura-Blaster gene knockout strategy targeting knockout and pCaEXP plasmid ectopic expression of mitochondrial respiratory function related gene MRF1,. The growth curve was determined by turbidimetric method, the sensitivity of MRF1 deficient bacteria to azolides and the tolerance of hydrogen peroxide stimulation were investigated by spot assay and micro liquid-based test. The mitochondrial membrane potential, ATP production and endogenous reactive oxygen species (Ros) of MRF1 gene deletion bacteria were detected by fluorescence dye. The changes of other known drug-resistance related genes were examined by real-time quantitative PCR. The changes of biofilm, mycelium formation ability, cell membrane sterol composition and efflux ability of MRF1 deficient bacteria were also investigated. The related functions of MRF1 gene were studied comprehensively. Conclusion: Candida albicans MRF1 gene deletion and high gene expression bacteria were successfully constructed, and after MRF1 gene deletion, mitochondrial membrane potential, ATP content and growth rate of the strain were slightly decreased, the sensitivity to oxidative stimulation was slightly increased, and ROS production was slightly increased. The Ybr026p encoded by MRF1 in Saccharomyces cerevisiae is 2-allythioate reductase, which catalyzes the last step reaction of mitochondrial fatty acid synthesis pathway 鈪，

本文編號(hào)：2262300