家蝇抗真菌肽-1A对人肝癌细胞HepG2抑制作用的机制  

Mechanism of inhibitory effect of MAF-1A on human hepatocellular carcinoma cell HepG2

作　　者：吴坤 马晓琳 张迎春 邓思波 黄敏慧 吴建伟 陈峥宏 王涛 WU Kun;MA Xiaolin;ZHANG Yingchun;DENG Sibo;HUANG Minhui;WU Jianwei;CHEN Zhenghong;WANG Tao(School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Medical Pathogen Biology of Education Department of Guizhou Province,Guiyang 550025,Guizhou,China;Jinan Jinyu Medical Test Center,Jinan 250000,Shandong,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 [3]济南金域医学检验中心,山东济南250000

出　　处：《贵州医科大学学报》2021年第2期131-136,142,共7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];贵州省科技计划项目[黔科合支撑(2020)4Y236]。

摘　　要：目的探究家蝇抗真菌肽-1A (MAF-1A)对人肝癌细胞Hep G2抑制作用的机制。方法采用原核串联表达及镍柱亲和层析法制备MAF-1A;取人肝癌细胞Hep G2进行传代培养,采用透射电镜技术,观察不同浓度MAF-1A(0、50、100 mg/L)作用细胞Hep G2细胞24 h后对细胞超微结构的影响;采用流式细胞术检测MAF-1A(0、100、200及400 mg/L)作用Hep G2细胞24 h后的细胞周期、细胞内ROS水平及细胞凋亡率;以荧光探针法检测MAF-1A(0、100、200及400 mg/L)作用Hep G2细胞24 h后的线粒体膜电位。结果透射电镜结果显示,MAF-1A作用后Hep G2细胞出现染色质边集,核固缩,胞膜出芽、空泡化、形成凋亡小体等形态学变化;流式细胞术检测结果显示,与0 mg/L MAF-1A组比较,MAF-1A各浓度组均可使G0/G1期的细胞比例升高(P<0.01),细胞凋亡率明显增高(P<0.01),当MAF-1A浓度达400 mg/L时、细胞内活性氧(ROS)含量明显增多(P<0.01);荧光探针检测发现,与0 mg/L MAF-1A组比较,MAF-1A可使Hep G2细胞的线粒体膜电位显著下降,并且随着MAF-1A浓度的升高,线粒体膜电位下降细胞数的占比增高。结论 MAF-1A可能通过促进线粒体膜电位下调、增加细胞内ROS含量所介导的细胞凋亡,以及阻滞细胞周期来发挥抑制Hep G2细胞的作用。Objective To explore the mechanism of Musca domestica antifungal peptide-1 A( MAF-1 A) on human hepatocellular carcinoma HepG2 cells. Methods MAF-1 A was prepared by tandem prokaryotic expression and nickel column affinity chromatography;human hepatocellular carcinoma cell HepG2 was subcultured,the effect of different concentrations of MAF-1 A( 0,50,100 mg/L) on the ultrastructure of HepG2 cells after 24 hours was detected by transmission electron microscopy;flow cytometry was used to detect the cell cycle,intracellular reactive oxygen species( ROS) level and apoptosis rate of HepG2 cells after treatment with of MAF-1 A( 0,100,200,and 400 mg/L) for24 h;the mitochondrial membrane potential of HepG2 cells treated with MAF-1 A( 0,100,200,and400 mg/L) for 24 h was detected by fluorescent probe method. Results HepG2 cells showed morphological changes such as chromatin edge set, karyopyknosis, budding of cell membrane,vacuolization and formation of apoptotic bodies after MAF-1 A treatment. Compared with the control group( 0 mg/L),the cell ratio of G0/G1 phase was increased in each MAF-1 A concentration group( P < 0. 01),and the apoptosis rate was significantly increased( P < 0. 01). When the concentration of MAF-1 A was 400 mg/L,the intracellular ROS level was significantly increased( P < 0. 01).Compared with the control group( 0 mg/L),MAF-1 A could significantly decrease the mitochondrial membrane potential of HepG2 cells,and with the increase of MAF-1 A concentration,the proportion of cells with decreased mitochondrial membrane potential increased. Conclusion MAF-1 A may play a role in inhibiting HepG2 cells by promoting cell apoptosis mediated by downregulation of mitochondrial membrane potential and increase of intracellular ROS content,as well as blocking cell cycle.

关 键 词：细胞凋亡 细胞周期 膜电位 线粒体 抗菌肽 家蝇抗真菌肽-1A 肝癌细胞 活性氧 

分 类 号：R735.7[医药卫生—肿瘤]