摘要
目的:进一步研究多药耐药1(multidrug resistance 1,MDR1)基因过表达在法尼醇促进氟康唑耐药白念珠菌凋亡过程中的机制。方法:从美国国家生物技术信息中心搜索获得MDR1基因的mRNA,利用双酶切法将MDR1基因的ORF插入质粒pCaEXP的MET3启动子后面,构建可过表达MDR1基因的pCaEXP-MDR1重组质粒。利用醋酸锂法将重组质粒转染至白念珠菌,制备MDR1过表达的白念珠菌作为实验组,转染pCaEXP空质粒的白念珠菌作为对照组。在2组中加入200μmol/L浓度的法尼醇培养后,采用四甲基偶氮唑盐(methylthiazolyl tetrazolium,MTT)比色法检测2组生长曲线,应用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)试剂盒检测并比较2组菌株的胱天蛋白酶3(caspase 3)的活化程度,菌株内谷胱甘肽(glutathione,GSH)的含量及细胞内活性氧类(reactive oxygen species,ROS)含量,进而比较不同组菌株在细胞凋亡、细胞内氧化还原及氧化应激水平上的差异。结果:与对照组比较,加入法尼醇后,MDR1过表达的实验组白念珠菌的繁殖生长曲线明显下降,其差异在24 h后有统计学意义(P=0.0392)。实验组细胞内还原型GSH的含量(P=0.0001)及氧化型GSH含量(P=0.0044)下降,ROS含量(P=0.0038)及胱天蛋白酶3活性(P=0.0290)上升。结论:MDR1表达的增加使法尼醇显著减少白念珠菌细胞内GSH的含量,增加细胞内ROS含量,细胞抗氧化能力下降,胱天蛋白酶3活性增加,促使白念珠菌凋亡。
Objective:To investigate the implication of overexpressed multidrug resistance 1(MDR1)gene in the farnesol promoted apoptosis of fluconazole resistant Candida albicans(C.albicans).Methods The sequence of MDR1 gene was retrieved from United States National Center for Biotechnology Information and its ORF was amplified and subcloned into a pCaEXP plasmid with MET3 promoter.The recombinant plasmid pCaEXP-MDR1 was transfected to C.albicans by lithium acetate to establish the MDR1 overexpressed strain,and C.albicans transfected with pCaEXP-empty plasmid was prepared as the control.The effect of farnesol(200μmol/L)exerated on the respective strains was examined with methylthiazolyl tetrazolium(MTT)assay.The caspase 3 activation,intracellular glutathione and reactive oxygen species(ROS)had also been determined with specific enzyme linked immunosorbent assay(ELISA)tests.Results Compared with control group,the growth curve of pCaEXP-MDR1 transfected C.albicans significantly decreased after 24 hours(P=0.0392).While intracellular glutathione levels dropped significantly in strains with MDR1 expression(P=0.0001),the levels of ROS(P=0.0038)and caspase 3 activity(P=0.0290)increased significantly.Conclusions MDR1 over-expression potentiates farnesol’s apoptosis promotion effect in C.albicans,by suppressing glutathione content,increasing ROS accumulation,decreasing cell antioxidant capacity and upregulating caspase 3 enzyme activity.
作者
康健
朱莹莹
张姣姣
王宁
王海嵘
周罗成
KANG Jian;ZHU Yingying;ZHANG Jiaojiao;WANG Ning;WANG Hairong;ZHOU Luocheng(Department of Emergency,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200096,China)
出处
《内科理论与实践》
2019年第6期366-370,共5页
Journal of Internal Medicine Concepts & Practice
基金
上海市卫生和计划生育委员会科研课题(项目编号:201540110)
上海市卫生计生系统重要薄弱学科建设计划(项目编号:2016zb0203)。
关键词
法尼醇
白念珠菌
多药耐药1基因过表达
Farnesol
Candida albicans
Multidrug resistance 1 gene over-expression
