细胞自噬对高三尖杉酯碱作用K562细胞的影响

摘要

Objective:To study the effect of homoharringtonine (HHT) alone or combined with 3-methyladenine (3 -MA),an autophagy inhibitor,on the apoptosis and autophagy of K562 cells.Methods:K562 cells were treated with HHT(10 ng/ml) or HHT(10 ng/ml) combined with 3-MA (1.5 mmol/L) for 1 to 8 days.The apoptosis of treated cells was tested by means of flow cytometry(FCM),and the autophagy levels were tested with RT-PCR,Western blot and electron microscopy.Results:In the early stage of HHT-treated group,the apoptosis rate increased and decreased later.Beclinl mRNA expression level and the LC3 Ⅱ/Ⅰ ratio were declined firstly and increased later in HHT group.While combining with autophagy inhibitor 3-MA,both the Beclinl mRNA expression level and the LC3 Ⅱ / Ⅰ ratio were declined continually during the treated period.The activated caspase-3 protein expression level was also raised sustainability during both HHT and 3-MA cultured period.Conclusions:HHT can induce apoptosis of K562 cells,but the sustaining effect of HHT can induc autophagy of K562 cells,the combination of HHT with 3-MA may enhance the cytotoxicitic effect of HHT on K562 cells.%目的:探讨高三尖杉酯碱(HHT)单用或与自噬抑制剂3-甲基腺嘌呤(3-MA)联用对K562细胞凋亡及自噬的影响.方法:K562细胞在HHT(10 ng/ml)作用下1-8d,采用流式细胞术检测细胞凋亡;采用RT-PCR法、Western blot法和透射电子显微镜检测细胞自噬.结果:在HHT作用前期K562细胞凋亡率逐渐升高,而在后期则下降.HHT作用后,K562细胞的Beclin1表达及LC3Ⅱ/Ⅰ蛋白比值先下降后升高.HHT单用时,激活型caspase-3蛋白表达前期逐渐上升,而在后期下降.与自噬抑制剂3-MA联用组的K562细胞的Beclin1表达和LC3Ⅱ/Ⅰ的比值持续下降,而激活型caspase-3蛋白表达则逐渐升高.结论:HHT可诱导K562细胞凋亡,但HHT持续作用可诱导K562细胞发生自噬,与3-MA联用则增强HHT的细胞毒作用.