KLF6基因对THP-1细胞诱导分化的巨噬细胞凋亡的影响

目的:探讨过表达Kruppel样因子6(KLF6)基因对THP-1细胞诱导分化的巨噬细胞活力、凋亡、活性氧簇(ROS)水平及AKT信号通路的影响。方法:使用佛波酯(PMA)将人单核细胞株THP-1诱导分化为巨噬细胞,将巨噬细胞随机分为pcDNA3.1组、氧化型低密度脂蛋白(ox-LDL)组、ox-LDL+pcDNA3.1组和ox-LDL+pcDNA3.1-KLF6组,其中pcDNA3.1转染参照Lipofectamine^TM 2000试剂盒说明。细胞处理24h,通过MTT实验、AnnexinV-FITC/PI双标细胞凋亡试剂盒和H2DCF-DA探针分别检测细胞活力、凋亡率和ROS水平的变化;Western blot检测Bcl-2、Bax和p-AKT的蛋白水平。结果:pcDNA3.1-KLF6转染巨噬细胞后,KLF6表达明显升高(P<0.05)。ox-LDL可明显抑制巨噬细胞的活力,诱导细胞凋亡,诱导细胞ROS的产生,上调Bax表达,下调Bcl-2和p-AKT的蛋白水平;而过表达KLF6可明显减弱ox-LDL对细胞活力、凋亡、ROS水平及Bcl-2、Bax和p-AKT蛋白水平的影响(P<0.05)。结论:KLF6基因可明显降低ox-LDL诱导的巨噬细胞凋亡,机制可能与降低细胞ROS水平及激活AKT信号通路有关。

Effect of KLF6 gene on apoptosis of THP-1 cell-derived macrophages

AIM:To investigate the effects of Kruppel-like factor 6(KLF6)over-expression on the viability,apoptosis,reactive oxygen species(ROS)level and AKT signaling pathway of THP-1 cell-derived macrophages.METHODS:Human monocyte cell line THP-1 was induced to differentiate into macrophages by phorbol myristate acetate(PMA),and the macrophages were randomly divided into pcDNA3.1 group,oxidized low-density lipoprotein(ox-LDL)group,ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-KLF6 group.pcDNA3.1 was transfected according to Lipofectamine TM 2000 Kit.The cell viability,apoptotic rate and ROS level were detected by MTT assay,flow cytometry with Annexin V-FITC/PI double staining and H 2DCF-DA probing,respectively.The protein levels of Bcl-2,Bax and p-AKT were determined by Western blot.RESULTS:After pcDNA3.1-KLF6 was transfected into the macrophages,the expression of KLF6 was increased significantly(P<0.05).ox-LDL significantly inhibited the viability of the macrophages,induced apoptosis and ROS production,up-regulated the protein expression of Bax,and down-regulated the protein levels of Bcl-2 and p-AKT(P<0.05).Over-expression of KLF6 significantly reduced the effects of ox-LDL on cell viability,apoptosis,ROS level and the protein levels of Bcl-2,Bax and p-AKT(P<0.05).CONCLUSION:KLF6 significantly reduces the apoptosis of THP-1 cell-derived macrophages induced by ox-LDL,which may be related to the reduction of ROS level and activation of AKT signaling pathway.