【摘要】 目的观察低氧和缺氧诱导因子-1α(HIF-1α)对K562细胞分泌血管相关生成因子的影响。方法选用携带和干扰HIF-1α基因的慢病毒转染K562细胞,以转染携带和干扰HIF-1α基因的K562细胞分别作为过表达组、干扰组,同时以空载病毒转染K562细胞作为对照组,3组在低氧状态下培养72h后收集细胞。通过荧光显微镜观察转染效率,采用RT-PCR法检测HIF-1α和血管相关生成因子mRNA表达水平;ELISA法检测培养上清液中血管相关生成因子蛋白水平。结果慢病毒转染K562细胞最佳感染复数(MOI)为10,转染效率约50%,经过嘌呤霉筛选后转染阳性细胞达90%以上。干扰组人血管生成素(ANG)-Ⅱ和血管内皮生长因子(VEGF)mRNA表达均低于过表达组,而转化生长因子(TGF)-βmRNA表达高于对照组和过表达组,过表达组ANG-ⅡmRNA表达高于对照组,差异有统计学意义(P<0.05)。培养上清液中TGF-α未检测到,过表达组ANG-Ⅱ水平低于对照组(P<0.05);过表达组VEGF和TNF-α水平高于对照组,干扰组TGF-β和VEGF水平低于对照组(P<0.05)。干扰组TGF-β、VEGF和TNF-α水平低于过表达组,差异有统计学意义(P<0.05)。结论低氧和HIF-1α可影响慢性白血病K562细胞血管生成相关因子的表达和自分泌,但在mRNA表达和蛋白分泌水平上存在差异。更多还原

【Abstract】 Objective To investigate the effect of hypoxia and hypoxia inducible factor 1α(HIF-1α)on human leukemia cell line K562 secreting angiogenesis-related factors.Methods The lentivirus carrying and interfering HIF-1αwas transfected into K562 cells.The K562 cells transfection carrying and interfering HIF-1αgene served as the overexpression group and interference group respectively.Meanwhile K562 cells with empty virus transfection served as the control group.The cells in 3 groups were harvested after 72 hof incubation under hypoxia.The transfection efficiency was observed by using the fluorescence microscope.The expression levels of HIF-1αand angiogenesis-related factors mRNA were detected by RT-PCR.The level of angiogenesisrelated factor protein in the culture supernatant was detected by ELISA.Results The optimal multiplicity of infection(MOI)of transfected K562 cells with lentivirus was 10 and the transfection efficiency was about50%.The transfection positive cells after screening by puromycin reached more than 90%.The expressions of ANG-Ⅱand VEGF mRNA in the interference group were all lower than those in the overexpression group,while the TGF-βmRNA expression was higher than that in the control group and overexpression group(P<0.05);the ANG-ⅡmRNA expression level in the overexpression group was higher than that in the control group,the difference was statistically significant(P<0.05).TGF-αin the culture supernatant fluid was not detected.The ANG-Ⅱlevel in the overexpression group was lower than that in the control group;the VEGF and TNF-αlevels in the overexpression group were higher than those in the control group,the TGF-βand VEGF levels in the interference group were lower than those in the control group(P<0.05).The levels of TGF-β,VEGF and TNF-αin the interference group were lower than those in the overexpression group.The above differences were statistically significant(P<0.05).Conclusion Hypoxia and HIF-1αcould affect the expression and autocrine of angiogenesis-related factors in chronic leukemia K562 cells.However,there are differences in mRNA expression and secretion levels.更多还原