【作者】 许懿；

【导师】 姜远英； 王彦；

【作者基本信息】 第二军医大学 ， 药理学， 2010， 博士

【摘要】 白念珠菌对氟康唑(FLC)的耐药性是临床抗真菌治疗失败的主要原因。本课题组前期的研究结果显示小檗碱(BBR)与FLC合用对耐药白念珠菌具有显著的协同抑菌作用。在本研究中,我们利用蛋白质二维电泳分析了临床耐药白念珠菌0304103、01010在FLC和BBR单用及合用前后的蛋白表达谱,所得结果经real-time RT-PCR验证,得到一批与能量代谢、氧化应激及其他功能相关的差异表达蛋白。此外,我们通过线粒体膜电位测定、细胞内ATP浓度测定、ATP合酶活性测定、内源性活性氧生成量测定、棋盘式微量液基稀释法考察还原剂的加入对两药协同效果的影响等实验进一步研究了BBR和FLC协同抗耐药白念珠菌的机制,结果显示两药合用后线粒体有氧呼吸增强,ATP生成量减少,ATP合酶活性受到抑制,内源性活性氧(ROS)生成大量增加,我们推断FLC与BBR合用是通过增强耐药白念珠菌的三羧酸循环、抑制ATP合酶活性使细胞内ROS生成大量增多,ROS通过氧化损伤作用杀伤耐药白念珠菌。这一“活性氧机制”可能是FLC与BBR协同抗耐药白念珠菌的重要分子机制之一。为深入研究BBR与FLC协同抗耐药白念珠菌的分子机制,我们利用cDNA芯片技术比较了临床耐药白念珠菌(0304103、01010、632)在FLC与BBR单用及合用前后的差异表达基因。通过对3株菌两药合用前后各自的差异表达基因取交集,共得到70个共同差异表达基因。所得差异表达基因均通过real-time RT-PCR进行验证。我们将70个共同差异基因进行了生物信息学分析及文献调研,发现两药合用后的差异表达基因主要与能量代谢、基因信息传递(复制、转录、翻译)及空泡形成三种生物学功能有关。其中,能量代谢相关基因的表达趋势与蛋白质组结果一致。根据芯片结果,我们推测:两药合用最终杀伤真菌细胞的核心机制可能是BBR的DNA损伤作用;白念珠菌可通过将BBR转运至其空泡内贮存从而起到对BBR的天然耐药作用。为探索“FLC + BBR”协同的关键环节,发现可能的“活性氧前”机制,根据蛋白质组及基因芯片结果提示,我们利用电镜及激光共聚焦显微镜观察BBR在白念珠菌细胞内的定位情况,发现BBR确实能贮存于空泡中并使空泡变大。通过琼脂点板实验,棋盘式微量液基稀释法及real-time RT-PCR等实验的结果提示,我们提出如下假说:白念珠菌可通过“囊泡运输”机制以胞吞的形式将BBR转运至空泡中贮存从而对其产生耐药现象;当合用FLC后,由于FLC的破膜作用可将空泡膜破坏从而释放出BBR,BBR的细胞毒作用导致胞内ROS生成大量增多,同时导致DNA损伤,进而而发挥抗耐药白念珠菌作用,该“空泡”机制可能是“FLC + BBR”协同抗耐药的关键环节。本课题为抗耐药白念珠菌的研究提供了新的思路。更多还原

【Abstract】 Multidrug resistance aggravates treatment failure of invasive infections with Candida albicans (C. albicans), a most common Candida pathogen. Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant C. albicans clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC (0304103, 01010) and the results were confirmed by real-time RT-PCR. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC+BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of anti-oxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC+BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans.To further reveal the specific synergistic mechanism of the combination of FLC and BBR against FLC-resistant C. albicans clinical strains, in this study, we performed a cDNA microarray analysis in untreated control cells and cells treated with FLC and/or BBR in 3 clinical strains of C. albicans resistant to FLC (0304103, 01010 and 632) and the results were confirmed by real-time RT-PCR. By taking overlap of differentially expressed genes identified in 3 clinical strains treated with or without FLC and/or BBR, our analyses identified 70 differentially expressed genes, 26 of which were function-unknown genes; 35 of which were related to multiple biochemical functions such as transcription regulator activity, translation regulator activity, RNA bingding, protein binding, cell cycle, cellular homeostasis, organelle organization, filamentous growth, transporter activity and multiple enzyme activity. After bioinformatics analysis and reading pertinent literature, we found that these function-known genes differentially expressed after the combnation treatment of FLC + BBR were mainly involved in 3 kinds of biochemical function: energy metabolisms, DNA damage and vacuolation. The expressed tendency of energy metabolisms-related genes were consistent with the results of the proteomic study performed previously. According the cDNA chip results, we speculated that the final mechanism to kill the Candida cells with the combination treatment of FLC + BBR is the DNA damage caused by BBR and there might be a natural BBR-resistant mechanism in C. albicans by storing BBR up in the vacuole.To explore the key mechanism of the synergistic action of FLC + BBR and to find the potential“ex-ROS”mechanism, based on the results of the proteomic and cDNA chip studies performed previously, we observed the location of BBR in C. albicans cells by uesing electron microscope and laser confocal microscopy. The results showed that the BBR was stored up in the vacuole and the vacuole were magnified after BBR treatment. In addition, further experiments including spot assay, checkerboard microdilution assay and real-time RT-PCR were performed. Based on the results of these experiments, we raised the hypothesis that C. albicans cells could store BBR up in the vacuole according to the“vesicle trafficing”mechanism, which caused the BBR-resistance in C. albicans. When combined with FLC to against C. albicans, BBR could released from the vacuole by the vacuolar membrane-disruptive effect of FLC and the cells were killed by the augmentation of ROS and DNA damage. This research provides a new direction to study multidrug-resistance in C. albicans.更多还原