家蝇抗真菌肽-1A体外抗白念珠菌的细胞内作用机制  

Intracellular mechanism of antifungal peptide-1A against Candida albicans in vitro

作　　者：邓思波 黄敏慧 张迎春 吴建伟 崔古贞 陈峥宏 王涛 DENG Sibo;HUANG Minhui;ZHANG Yingchun;WU Jianwei;CUI Guzhen;CHEN Zhenghong;WANG Tao(School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]贵州省普通高等学校病原生物学特色重点实验室,贵州贵阳550025

出　　处：《贵州医科大学学报》2021年第12期1365-1369,共5页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81360254);贵州省科技计划项目[黔科合平台人才(2018)5799-22,黔科合支撑(2020)4Y236]。

摘　　要：目的探讨家蝇抗真菌肽-1A(MAF-1A)体外抗白念珠菌(C.albicans)的细胞内作用机制。方法取C.albicans冻存物划线接种、培养并制备浓度为2×10^(9) cfu/L的菌悬液;分别以0、625、1250及2500 mg/L的MAF-1A作用24 h,采用荧光指示剂法检测各处理细胞内活性氧(ROS)含量的变化;取0、781及1560 mg/L抗氧化剂维生素C(V_(c))和还原型谷胱甘肽(GSH))预处理C.albicans细胞30 min,采用微量稀释法检测MAF-1A抗C.albicans的最小抑菌浓度(MIC);采用荧光指示剂法检测0、625、1250及2500 mg/L的MAF-1A作用24 h后C.albicans细胞线粒体膜电位的变化;取C.albicans脱氧核糖核酸(DNA)与等体积不同浓度(0、625、1250及2500 mg/L)MAF-1A共孵育6 h,采用琼脂糖凝胶阻滞实验分析MAF-1A对C.albicans DNA的影响。结果与0 mg/L MAF-1A相比,不同浓度MAF-1A处理C.albicans细胞内ROS量明显增加(P<0.01),且胞内ROS的含量随着MAF-1A处理浓度的增加而增加,呈剂量依赖性;V C和GSH处理后,MAF-1A抗C.albicans的MIC值由625 mg/L升高至1250 mg/L;MAF-1A处理后,C.albicans细胞线粒体的膜电位未出现变化(P>0.05);凝胶阻滞实验结果显示,不同浓度MAF-1A与C.albicans DNA孵育后,DNA条带出现滞后及变暗淡现象,且在加样孔有滞留,这种阻滞现象随着MAF-1A浓度升高越明显。结论MAF-1A不仅能诱导C.albicans细胞内大量产生ROS,还能通过与DNA的结合而发挥对C.albicans的抑制作用。Objective To explore the intracellular mechanism of Musca domestica antifungal peptide-1A(MAF-1A)against Candida albicans in vitro.Methods C.albicans frozen deposits were underlined to inoculate,cultured,and prepared at a concentration of 2×10^(9) cfu/L bacterial suspension.At 0,625,1250,and 2500 mg/L MAF-1A for 24 h,the cellular content of reactive oxygen species(ROS)in C.albicans was determined by fluorescence indicator method.With 0,781,and 1560 mg/L antioxidant vitamin C(V_(c))and also the prototype glutathione(GSH))pretreatment with C.albicans 30 min,the minimum antibacterial concentration(MIC)of the MAF-1A antibody against C.albicans was measured by microdilution.The changes of mitochondrial membrane potential in C.albicans cells after 24 h of 0,625,1250,and 2500 mg/L MAF-1A were determined by a fluorometric indicator assay.The incubation of deoxyribonucleic acid(DNA)with C.albicans was MAF-1A at different concentrations(0,625,1250,and 2500 mg/L)for 6 h,and the effect of MAF-1A on C.albicans DNA was analyzed by agarose gel block assay.Results ROS in C.albicans increased significantly at different MAF-1A concentrations(P<0.01),and the content of intracellular ROS increased dose-dependent with MAF-1A concentration.After V C and GSH treatment,the MIC value of MAF-1A against C.albicans was increased from 625 mg/L to 1250 mg/L.No membrane potential was changed in the C.albicans cell mitochondria after treatment with MAF-1A(P>0.05).The results of the gel retardation experiments showed that the DNA bands were lagging and dim after incubation of different concentrations of MAF-1A with C.albicans DNA.There was retention in the additive hole,and this arrest was more obvious with the increase of MAF-1A concentration.Conclusion MAF-1A can not only induce a massive production of ROS in C.albicans cells,but also exert an inhibitory effect on C.albicans by binding to DNA.

关 键 词：抗菌肽类 念珠菌 白色 抗氧化剂 膜电位 线粒体 家蝇抗真菌肽-1A 活性氧 DNA凝胶阻滞 作用机制 

分 类 号：R735.7[医药卫生—肿瘤]