铁过载抑制人肝癌Huh7.5细胞增殖和促进细胞凋亡的机制

发布时间：2018-03-29 05:33

  本文选题：慢性丙型肝炎　切入点：铁过载　出处：《细胞与分子免疫学杂志》2017年08期

【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。
[Abstract]:Objective to observe the effect of iron overload on the activity of human hepatoma Huh7.5 cells and its mechanism. Methods Huh7.5 cells were treated with 50 100 200 渭 mol/L ferric ammonium citrate (FAC). The ferric ion fluorescence probe Phen Green FL labeling and fluorescence microscope were used to detect the iron load of the cells and the proliferation activity of the iron overload cells. Real-time quantitative PCR was used to detect the levels of TfR2, divalent metal ion transporter 1 (DMT1) and membrane iron transporter 1 (FPN1) in iron-overload cells. Western blot was used to detect the levels of TfR1TfR2DMT1 and FPN 1 protein. DCFH-DAA staining combined with flow cytometry (FCM) was used to detect the level of reactive oxygen species (Ros) in cells treated with FAC and FAC combined with 400 渭 mol/L N-acetylcysteine (NAC). Apoptosis was detected by double staining of annexin 鈪，

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