法尼醇促氟康唑耐药白念珠菌死亡的实验研究

Experimental study on farnesol induced death of fluconazole resistant Candida albicans

目的:初步探讨法尼醇促进氟康唑耐药白念珠菌死亡的可能机制。方法 :使用药物浓度梯度法构建氟康唑耐药白念珠菌;采用实时定量PCR技术检测耐药、敏感白念珠菌中耐药相关基因的表达。四甲基偶氮唑盐(MTT法)比色法检测法尼醇对氟康唑耐药、敏感白念珠菌增殖的影响。应用流式细胞技术及激光共聚焦显微镜技术,比较2组菌株在细胞凋亡、坏死及细胞内氧化应激水平上的差异。结果:成功构建了氟康唑耐药白念珠菌菌株,氟康唑耐药白念珠菌中多药耐药基因1(MDR1)的表达较敏感株明显增加(P<0.01);而白念珠菌耐药(CDR)1、CDR2基因的表达在2组中并无明显差异(P>0.05)。与氟康唑敏感白念珠菌相比,法尼醇介导耐药白念珠菌细胞内活性氧类(ROS)积聚增多,并显著促进耐药白念珠菌发生坏死。结论 :MDR1基因在氟康唑耐药白念珠菌中高表达;法尼醇可介导氟康唑耐药白念珠菌细胞内ROS积聚增多,并促进耐药菌株发生坏死。

Objective To investigate the possible mechanism of farnesol induced death of fluconazole resistant Candida albicans. Methods Drug concentration gradient method was used to construct fluconazole resistant Candida albicans.The expression of multi-drug resistance（MDR） genes was detected by real-time quantitative PCR. Dimethyl thiazol diphenyl tetrazolium bromide（MTT） assay was used to detect the effect of farnesol on fluconazole resistant/sensitive Candida. Strain difference was compared in cell apoptosis,necrosis and reactive oxygen species（ROS） level by using flow cytometry and laser confocal microscopy. Results Fluconazole resistant Candida albicans strains were successfully constructed,and expression of MDR1 gene was significantly increased in these strains（P〈0.01）,while there was no significant increase in expression of Candida drug resistance（CDR）2 and CDR1 gene. Compared with fluconazole sensitive Candida albicans,farnesol mediated the accumulative increase of ROS in fluconazole resistant Candida albicans,and it significantly promoted the necrosis of fluconazole resistant Candida albicans. Conclusions The MDR1 gene was overexpressed in fluconazole resistant Candida albicans; farnesol increased ROS accumulation and promoted the necrosis of fluconazole resistant Candida albicans.