氟康唑诱导热带念珠菌凋亡机制的研究

Study of the apoptosis mechanism of fluconazole in Candida tropicalis

目的观察氟康唑对热带念珠菌作用后存活率、活性氧(ROS)、线粒体膜电位(△Ψm)和细胞周期的变化,初步探讨其作用机制。方法参照CLSIM27A方案中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);热带念珠菌在加入不同浓度氟康唑培养后,流式细胞术(FCM)分析荧光探针碘化丙啶(PI)标记存活率、荧光探针双氢罗丹明(DHR)标记ROS、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△Ψm和DNA-Prep试剂盒标记细胞周期的变化。结果氟康唑作用后,热带念珠菌氟康唑耐药株存活率、ROS、线粒体△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存活率和线粒体△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,G01期热带念珠菌减少,呈一定的时间剂量依赖关系;且ROS升高与线粒体△Ψm成正相关(r=0.913,P【0.05)。结论氟康唑可能提高刺激后热带念珠菌产生ROS,导致线粒体△ψm的改变和G2/M阻滞,从而诱导热带念珠菌凋亡。

Objective To investigate the changes of viability rate、 reactive oxygen species(ROS)、 mitochondrial membrane potential (△Ψm) and cell cycle of Candida tropicalis after the treatment with fluconazole and to explore its effect mechanism.Methods The MICs of the clinical isolates Candida tropicalis to fluconazole were tested by M27-A microdilution method.Candida tropicalis stains were incubated with different concentrations fluconazole.Viability rate was measured by PI staining,the intracellular accumulation of ROS was determined by dihydrohodamine 123 (DHR) staining,the loss of mitochondrial △Ψm was detected with 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetrethyl benzimidalyl carbocyanine iodide( JC-1) staining and cell cycle was determined by DNA-Prepkit by flow cytometry,respectively.Results Viability rate、 ROS 、mitochondrial △Ψm and cell cycle in Candida tropicalis fluconazole resistance stains were no significant variation,but a decrease of mitochondrial △Ψm and viability rate、 an increase of ROS accumulation in Candida tropicalis fluconazole susceptibile stains were detected in a time-dose-dependent manner; a majority of Candida tropicalis were arrested in G2/M phase and a progressive decline of Candida tropicalis was seen in G01.There were strong associations between ROS accumulation and change of mitochondrial△Ψm at corresponding concentration points(r=0.913,P<0.05).Conclusion Fluconazole induced intracellular accumulation of ROS,which could resulte in apoptosis of Candida tropicalis because of change of mitochondrial △Ψm and G2/M arrest.