發(fā)布時間：2018-01-21 11:16

  本文關(guān)鍵詞： 白念珠菌 線粒體 基因敲除 生物被膜 內(nèi)源性活性氧　出處：《第二軍醫(yī)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：白念珠菌易于感染、難以防治的特點使其在臨床治療中表現(xiàn)為高致病性和高耐藥性。目前國際上對白念珠菌基因組的測序工作已經(jīng)完成,并可以通過公共數(shù)據(jù)庫可以獲得相應(yīng)的完整基因組序列。經(jīng)過DNA序列及編碼的氨基酸序列比對,發(fā)現(xiàn)一個白念珠菌新基因IPF19998與釀酒酵母中的AIF1基因有高度同源性。AIF1是與人類AIF同源的酵母細胞凋亡誘導(dǎo)因子(Apoptosis inducing factor)。已經(jīng)證實在釀酒酵母中,在由H2O2或醋酸鹽誘發(fā)的凋亡以及衰老過程中,Aif1p從線粒體中轉(zhuǎn)位至細胞核內(nèi),并誘導(dǎo)細胞凋亡。在AIF1基因敲除菌株中,凋亡特征消失,衰老延緩[1]。由此我們推測,IPF19998是白念珠菌中的凋亡誘導(dǎo)因子,并在白念珠菌細胞凋亡過程中發(fā)揮重要作用,對該基因的進一步研究有助于闡明白念珠菌細胞凋亡機制,為抗真菌感染藥物的研究設(shè)計提供新思路。 本課題采用Ura-Blaster基因敲除策略靶向敲除和pCaEXP質(zhì)粒異位表達凋亡誘導(dǎo)因子基因IPF19998,構(gòu)建了IPF19998基因缺失菌和高表達菌。將構(gòu)建好的菌株通過比濁法測定生長曲線;通過微量液基實驗來考察IPF19998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;將缺失菌在塑料培養(yǎng)板中按照生物被膜形成條件培養(yǎng)24小時并形成生物被膜,考察其生長動力學(xué)變化;通過熒光染料檢測IPF19998基因缺失菌在酵母態(tài)及被膜態(tài)兩種狀態(tài)下的內(nèi)源性活性氧水平(ROS),通過實時定量PCR考察了IPF19998基因缺失后,其他已知氧化還原相關(guān)基因和生物被膜形成相關(guān)基因表達量的變化。結(jié)論:成功構(gòu)建白念珠菌IPF19998基因缺失菌和基因高表達菌株;IPF19998基因缺失后,菌株生長速度不變;但對唑類藥物敏感性沒有明顯變化,高表達菌株對唑類藥物產(chǎn)生耐藥性;對過氧化氫刺激所引起的凋亡也與野生菌相比,無明顯差異。這與我們前期設(shè)想的結(jié)論不同。但用熒光染料測其內(nèi)源性活性氧水平(ROS),與酵母態(tài)下生成的ROS含量相比,被膜態(tài)下的菌株,不論是敲除菌株,高表達菌株還是野生型菌株,其ROS產(chǎn)生遠遠低于酵母態(tài)下的ROS產(chǎn)生。而在被膜態(tài)下,IPF19998基因缺失菌ROS產(chǎn)生增多,而高表達菌ROS產(chǎn)生保持一個穩(wěn)定且低的水平。同時,我們發(fā)現(xiàn),IPF19998基因缺失以后,其形成生物被膜的能力下降,高表達菌株生成生物被膜的能力明顯增強。這一結(jié)果在Real-time實驗中也得到證實。IPF19998基因能改變已知氧化還原相關(guān)基因和生物被膜形成相關(guān)基因表達量的變化。根據(jù)本研究推測,IPF19998基因可通過增強抗氧化能力,清除細胞內(nèi)的ROS含量,增強生物被膜的形成能力,從而降低對藥物的敏感性。與預(yù)測的促進細胞凋亡功能無關(guān),而與生物被膜形成過程中的抗氧化能力有關(guān)。具體機制有待進一步探討。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R379

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本文編號：1451327