HIF-1α对K562细胞血管生成相关因子的影响  

Effect of HIF-1α on Angiogenesis-Related Factors in K562 Cells

作　　者：解友邦[1] 李建平[1] 沈括[1] 孟芳[1] 王莉[1] 韩国雄[1] 艾国[1] 蒋白丽 赵强强[1] 侯艳 杨红艳[1] 李文倩[1] XIE You-Bang;U Jian-Ping;SHENG Guo;MENG Fang;WANG Li;HAN Guo-Xiong;AI Guo;JIANG Bai-Li;ZHAO Qiang-Qiang;HOU Yan;YANG Hong-Yan;LI Wen-Qian(Department of Hematology,People's Hospital of Qinghai Province,Xining 810007,Qinghai Province,China)

机构地区：[1]青海省人民医院血液风湿科

出　　处：《中国实验血液学杂志》2019年第5期1476-1481,共6页Journal of Experimental Hematology

基　　金：青海省卫生和计划生育委员会2016年科研指导项目(青卫科2016-22);青海省国家重点专科项目(卫办医政函2011-873);2014年度青海省人才小高地项目(青海人才字2014-12);青海省高层次卫生人才(骨干人才和领军人才)计划

摘　　要：目的:通过在K562细胞过表达和干扰HIF-1α后检测K562细胞分泌血管生成相关因子的水平,探讨慢性白血病血管生成的机制。方法:应用携带和干扰HIF-1α基因的慢病毒转染K562细胞,将转染携带和干扰HIF-1α基因的K562细胞分别纳入过表达组、干扰组,同时以空载病毒转染K562细胞纳入对照组。3组细胞在常氧状态下培养72 h后收集细胞,通过荧光显微镜观察3组转染效率,采用RT-PCR法检测HIF-1α和血管相关生成因子mRNA表达水平,ELISA法检测培养上清中血管相关细胞因子的浓度。结果:慢病毒转染K562细胞最佳转染复数(MOI)为10,转染效率为50%左右;经过嘌呤霉筛选后转染阳性细胞达90%以上。干扰组ANG-I、ANG-II、TGF-α和VEGF mRNA表达均低于过表达组,而干扰组TGF-β1 mRNA表达高于过表达组。干扰组ANG-I、VEGF mRNA表达低于对照组;培养上清中未检测到TGF-α,干扰组ANG-I和TNF-α水平低于过表达组,而干扰组VEGF水平高于过表达组;过表达组和干扰组TGF-β1水平均低于对照组,过表达组VEGF水平低于对照组,干扰组VEGF水平高于过表达组和对照组,上述差异具有统计学意义(P<0.05)。结论:通过嘌呤霉素筛选获得慢病毒转染K562细胞阳性细胞,HIF-1αmRNA正向调控K562细胞ANG-1、ANG-2、TGF-α、VEGF mRNA的表达,促进ANG-I和TNF-α自身分泌的能力,而不刺激TGF-α的自分泌,HIF-1α上调可抑制K562细胞TGF-β1的表达并抑制TGF-β的自分泌。Objective:To explore the mechanisms of angiogenesis in chronic myeloid leukemia(CML)through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1αgene in K562 cells.Methods:The K562 cells were transfected by lentiviruses carried and interfered HIF-1αgene,then the transtected K562 cells with carried and interfered with HIF-1αgene were enrolled in overexpression and interference groups respectively,at the same time the K562 cells transfected by the empty virus were enrolled in control group.The cells were harvested after culture for 72 hours under normoxid condition.The transfection efficient in 3 groups was detected by fluorescence microscopy;the mRNA expression of HIF-1αgene and angiogenesis-related factors was detected by RT-PCR;the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA.Results:The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%.The positive rate of transfection after screening by puromycin was more than 90%.The mRNA expression of ANG-I,ANG-II,TGF-αand VEGF in the interference group was lower than that in the over-expression group,and the TGF-β1 mRNA expression in the interference group was higher than in the over-expression group.The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group.TGF-αdid not could be detected,and the culture supernatant concentration of ANG-I and TNF-αin the interference group was lower than in the over-expression group,while the VEGF concentration in the interference group was higher than that in the overexpression group.All of the above-mentioned differences were statistically significant(P<0.05).Conclusion:The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin.The HIF-1αmRNA positively regulates the mRNA expression of ANG-1,ANG-2,TGF-α,VEGF in K562 cells,promotes the antocrine ability of ANG-1 and T

关 键 词：K562细胞 HIF-1Α 血管生成相关因子 

分 类 号：R733.72[医药卫生—肿瘤]