家蝇抗真菌肽-1A对人肝癌细胞HepG2的影响  

Effect of MAF-1A on human hepatocellular carcinoma cell HepG2

作　　者：吴坤 张迎春 邓思波 黄敏慧 吴建伟 陈峥宏 王涛 WU Kun;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)

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2期125-130,142,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：目的探讨家蝇抗真菌肽-1A(MAF-1A)对人肝癌细胞Hep G2的作用。方法取人肝癌细胞Hep G2和人正常肝细胞LO2进行传代培养;采用原核串联表达及镍柱亲和层析法制备MAF-1A,采用显微观察法检测0、50及100 mg/L MAF-1A作用Hep G2细胞24 h后的细胞形态变化,采用噻唑蓝(MTT)法检测0、25、50、100、200、400及800 mg/L MAF-1A分别作用于Hep G2和LO2细胞24 h后的细胞生存率,采用乳酸脱氢酶(LDH)释放试验检测0、25、50、100、200及400 mg/L MAF-1A作用Hep G2细胞24 h后对细胞的破坏性,采用细胞划痕愈合试验检测0、100及200 mg/L MAF-1A作用Hep G2细胞12和24 h后细胞迁移能力的变化;用磷酸盐缓冲液(PBS)将抗凝的人红细胞浓度调节为1%体积分数,以1%Triton X-100为阳性对照组,PBS为阴性对照组,采用红细胞体外溶解试验检测100、200、400、800、1200、1 800及2 400 mg/L MAF-1A作用人红细胞1 h后的溶血活性。结果 MAF-1A作用24 h后,镜下可见Hep G2细胞出现表面皱褶、坏死脱落等细胞病变效应(CPE),且浓度越高、CPE越明显;与0 mg/L组比较,各浓度组MAF-1A均对Hep G2细胞有抑制作用(P<0.05或P<0.01),并呈浓度依赖性,但对LO2细胞的抑制作用不明显;MAF-1A使细胞释放LDH的量增多,且LDH释放率随着MAF-1A浓度的升高而增大(P<0.01);各浓度组MAF-1A均能抑制Hep G2细胞的迁移运动,高浓度组抑制作用更为明显(P<0.05);MAF-1A浓度达2 400 mg/L时,溶血率<1%。结论 MAF-1A能抑制Hep G2细胞的增殖及迁移,但对人正常肝细胞LO2和红细胞无明显毒性作用。Objective To explore the effect of Musca domestica antifungal peptide-1 A( MAF-1 A) on human hepatocellular carcinoma cell HepG2. Method Human hepatocellular carcinoma cell HepG2 and human normal liver cells LO2 were subcultured. MAF-1 A was prepared by tandem prokaryotic expression and nickel column affinity chromatography. The effect of different concentrations of MAF-1 A( 0,50,100 mg/L) on the morphology of HepG2 cells in 24 h was detected by microscopic observation. MTT assay was used to detect the survival rate of HepG2 and LO2 cells treated with MAF-1 A( 0,25,50,100,200,400,and 800 mg/L) for 24 h,respectively. The damage to HepG2 cells24 h after action by MAF-1 A( 0,25,50,100,200,and 400 mg/L) was detected by LDH release test. Cell scratch healing test was used to detect the effect of MAF-1 A( 0,100,200 mg/L) on the migration ability of HepG2 cells in 12 h or 24 h. The concentration of anticoagulant human red blood cells was adjusted to 1% volume fraction with PBS,Triton X-100 as the positive control group and PBS as the negative control group,after 1 h treatment with MAF-1 A( 100,200,400,800,1 200,1 800,and 2 400 mg/L),hemolysis activity was detected by extracorporeal erythrocyte dissolution test.Result After treatment with MAF-1 A for 24 h,HepG2 cells showed CPE such as surface wrinkles and necrosis,and the higher the concentration,the more obvious the CPE was. Compared with 0 mg/L MAF-1 A group,the results of MTT assay showed that MAF-1 A had a strong inhibitory effect on the growth of HepG2 cells in a dose-dependent manner( P < 0. 05 or P < 0. 01),but had no obvious inhibitory effect on LO2 cells. MAF-1 A increased the amount of LDH released by the cells,and the release rate of LDH increased with the increase of MAF-1 A concentration( P < 0. 01). MAF-1 A could inhibit the migration of HepG2 cells in all concentration groups,especially in high concentration group( P < 0. 05). When the concentration of MAF-1 A reached to 2 400 mg/L,the hemolysis rate was still less than 1%. Conclusion MAF-1 A can inhibit

关 键 词：细胞迁移分析 抗菌肽 家蝇抗真菌肽-1A 肝癌细胞 溶血性 抗肿瘤活性 

分 类 号：R735.7[医药卫生—肿瘤]