铁过载抑制人肝癌Huh7.5细胞增殖和促进细胞凋亡的机制  

Mechanism for the inhibition of proliferation and promotion of apoptosis in Huh7.5 cells by iron overload

作　　者：秦源[1] 卫静[2] 张怡 康玲[4] 亢必勃[1] 申焕君[1] 郑煦暘[1] 王亚宁[1] 贾战生[1] 张颖[1] QIN Yuan WEI Jing ZHANG Yi KANG Ling KANG Bibo SHEN Huanjun ZHENG Xuyang WANG Yaning JIA Zhansheng ZHANG Ying(Department of Infectious Diseases Department of Endocrinology, Tangdu Hospital, Fourth Military Medical University, Xi ＇an 710038 Xi＇an Radios ＆ Television University, Xi ＇an 710061 Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi＇an 710038, China)

机构地区：[1]第四军医大学唐都医院全军感染病诊疗中心,陕西西安710038 [2]第四军医大学唐都医院内分泌科,陕西西安710038 [3]西安广播电视大学,陕西西安710061 [4]第四军医大学唐都医院妇产科,陕西西安710038

出　　处：《细胞与分子免疫学杂志》2017年第8期1056-1061,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81273218)

摘　　要：目的观察铁过载对人肝癌Huh7.5细胞活性的影响及机制。方法用(50、100、200)μmol/L枸橼酸铁铵(FAC)处理Huh7.5细胞,铁离子荧光探针Phen Green FL标记结合荧光显微镜检测细胞铁载量。MTT法检测铁过载细胞的增殖活性,实时定量PCR检测铁过载细胞运铁蛋白受体1(TfR1)、TfR2、二价金属离子转运体1(DMT1)和膜铁转运蛋白1(FPN1)的mRNA水平,Western blot法检测TfR1、TfR2、DMT1和FPN 1蛋白水平,二氯二氢荧光素乙酰乙酸酯(DCFH-DA)染色结合流式细胞术检测活性氧(ROS)水平;在FAC处理细胞以及FAC联合400μmol/L N-乙酰半胱氨酸(NAC)处理后,采用异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染色结合流式细胞术检测细胞凋亡。结果细胞铁载量随FAC增加呈浓度依赖性上升;FAC处理细胞TfR1、TfR2和DMT1 mRNA和蛋白水平显著下调,FPN1 mRNA和蛋白水平显著上调;ROS水平随FAC浓度增加显著升高,细胞增殖活性随FAC浓度增加显著下降;FAC处理细胞的凋亡率均显著高于对照组,而向FAC处理细胞加入抗氧化剂NAC后,细胞凋亡率显著下降。结论铁过载抑制Huh7.5细胞增殖活性并促进细胞凋亡,其作用机制可能与氧化应激途径有关。Objective To investigate the effect of iron overload on biological activity and apoptosis in Huh7. 5 cells.Methods Huh7. 5 cells were cultured in the medium supplemented with 50,100,200 μmol/L ferric ammonium citrate（ FAC）.Fluorescence microscopy was employed to determine cel iron load labeled by Phen Green FL; proliferation activity of Huh7. 5cel s was evaluated by MTT assay; protein and mRNA levels of transferrin receptor（ TfR1）,TfR2,divalent metal transporter 1（ DMT1） and ferroportin 1（ FPN1） in Huh7. 5 cells were detected by Western blotting and real-time PCR,respectively; cell reactive oxygen species（ ROS） labeled by dichlorofluorescin diacetate（ DCFH-DA） and cell apoptosis labeled by annexinⅤ-FITC/PI were analyzed by flow cytometry. Results FAC treatment increased intracellular iron load in a dose-dependent manner. Compared with control group,mRNA and protein expressions of TfR1,TfR2 and DMT1 were down-regulated,while mRNA and protein expression of FPN1 was significantly up-regulated in FAC treated groups. With the increasing dose of FAC,intracellular ROS level increased significantly and cell proliferation activity decreased significantly. The cell apoptosis rate in FAC treated groups were remarkably higher than that in control group,but after antioxidant N-acetylcysteine（ NAC）was added,the cell apoptosis in FAC treated group was inhibited obviously. Conclusion Iron overload can inhibit the proliferation and promote the apoptosis of Huh7. 5 cells through oxidative stress.

关 键 词：慢性丙型肝炎 铁过载 细胞增殖活性 细胞凋亡 活性氧(ROS) 

分 类 号：R512.6[医药卫生—内科学] Q279[医药卫生—临床医学]