氟康唑对光滑假丝酵母菌及线粒体膜电位的影响

Effect of Fluconazole on Mitochondrial Membrane Potential and Candida glabrata

目的观察氟康唑对光滑假丝酵母菌作用后存活率、细胞周期、活性氧(ROS)和线粒体膜电位的变化,初步探讨其作用机制。方法采用NCCLS M27-A微量稀释法和ATBF3法测定临床分离的光滑假丝酵母菌对氟康唑的最低抑菌浓度(MIC)值;光滑假丝酵母菌氟康唑敏感株与光滑假丝酵母菌氟康唑耐药株与不同浓度氟康唑共同培养后,流式细胞术(FCM)分析荧光探针碘化丙啶(PI)标记存活率、DNA-Prepkit试剂盒标记细胞周期、荧光探针双氢罗丹明(DHR)标记ROS和荧光探针四氯四乙基苯并咪唑基羰花青碘化物(JC-1)标记线粒体膜电位的变化。结果氟康唑敏感株MIC在2μg/ml,氟康唑耐药株MIC>128μg/ml;氟康唑作用后,氟康唑耐药株存活率、细胞周期、ROS和线粒体膜电位均无明显变化;而氟康唑敏感株存活率明显下降,ROS和线粒体膜电位明显升高,呈一定的剂量依赖关系;大部分光滑假丝酵母菌阻滞于G2-M期,并出现subG1凋亡峰。结论光滑假丝酵母菌过多ROS的产生和线粒体膜电位去极化可能是氟康唑诱导光滑假丝酵母菌凋亡的重要机制。

OBJECTIVE To investigate the changes in viability rate,reactive oxygen species（ROS）,cell cycle and mitochondrial membrane potential after treatment with fluconazole to Candida glabrata,and to explore its effect mechanism.METHODS The MICs of the clinical isolates of C.glabrata to fluconazole were tested by M27-A microdilution method and ATBF3 method.The fluconazole-resistant and-sensitive C.glabrata strains were incubated with different concentrations of fluconazole and their,viability rate was measured by propidium iodide（PI） staining.The cell cycle was determined by DNA-Prepkit,the intracellular accumulation of ROS was determined by dihydrorhodamine 123（DHR） staining and the loss of mitochondria was detected with 5,5,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidalyl carbocyanine iodide（JC-1） staining by flow cytometry,respectively.RESULTS After treatment with fluconazole,the viability rate,cell cycle,ROS and mitochondrial membrane potential in fluconazole-resistant C.glabrata strains showed no significant difference,but in fluconazole-sensitive C.glabrata strains the viability rate decreased and the ROS accumulation and mitochondrial membrane potential increased in a dose-dependent manner.A majority of C.glabrata strains were arrested in G2/M phase and the apoptosis peak appeared in sub-G1.CONCLUSION Fluconazole induced apoptosis of C.glabrata is probably related to the intracellular accumulation of ROS and the change in mitochondrial membrane potential.