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为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,△Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC);热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化;而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降,ROS明显升高,而且大部分热带念珠菌阻滞于G2/M期,并出现明显凋亡峰,呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生,阻止细胞周期G2/M期阻滞和降低凋亡。由此可见,氟康唑可能通过刺激热带念珠菌产生过多ROS,并使线粒体膜电位△Ψm下降,从而诱导热带念珠菌凋亡。
To investigate the mechanism of action of fluconazole, the survival rate, the change of reactive oxygen species (ROS), mitochondrial membrane potential (△ Ψm) and cell cycle of Candida tropicalis were observed. The minimal inhibitory concentration (MIC) of fluconazole against Candida tropicalis was determined by microdilution method according to NCCLS M27-A protocol. Flow cytometry (FCM) was used to detect the MIC of Candida tropicalis and different concentration of fluconazole. Candida tropicalis survival rate, ROS, mitochondrial membrane potential △ Ψm and cell cycle changes were analyzed. The results showed that after fluconazole treatment, the survival rate of fluconazole-resistant strains of Candida tropicalis, ROS, mitochondrial membrane potential △ Ψm and the proportion of cell cycle stages did not change significantly; while tropical Candida fluconazole sensitive strains Survival rate and mitochondrial membrane potential △ Ψm decreased significantly, ROS increased significantly, and most of the tropical Candida block G2 / M phase, and the apparent peak of apoptosis, showed a time-dependent manner. Free radical scavenger glutathione inhibits the production of Candida tropicalis ROS, arrests the G2 / M phase of the cell cycle and reduces apoptosis. Thus, fluconazole may induce excess Candida tropicalis by stimulating the Candida tropicalis and decreasing the mitochondrial membrane potential △ Ψm.