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目的探讨过氧化物酶体增殖物激活受体γ(PPARγ)激动剂匹格列酮(PGZ)对白血病HL-60细胞的体外增殖抑制作用及其作用机制。方法以不同浓度的PGZ(20～80μmol/L)作用于体外培养的HL-60细胞24h、48h及72h,MTT法检测细胞生长抑制率,流式细胞术(FCM)检测细胞周期及细胞凋亡。应用RT-PCR及免疫印迹法(Westernblot)检测药物作用后PPARγ、Caspase-3及其裂解底物多聚(ADP-核糖)聚合酶(PARP)表达水平的变化。结果 PGZ可显著抑制细胞的生长及诱导细胞发生凋亡,呈现出明显的量-效与时-效关系。FCM检测结果表明,细胞主要被阻滞在G0/G1期,60μmol/L的PGZ作用48h后可以出现典型的亚G1期峰(细胞凋亡峰),PGZ在诱导细胞凋亡的同时,PPARγmRNA及蛋白的表达水平均逐渐升高。RT-PCR表明,Caspase-3酶原及其作用底物PARP表达水平逐渐降低,Westernblot结果显示32kD的Caspase-3酶原被活化出现17kD亚单位片段,同时Caspase-3的底物PARP被裂解出现89kD的亚单位片段。结论 PGZ在体外对HL-60细胞具有显著的细胞周期阻滞作用,并诱导细胞发生调亡。通过依赖PPARγ信号途径以及激活Caspase-3可能是PGZ抑制细胞增殖及诱导细胞发生凋亡的重要作用机制之一。
Objective To investigate the inhibitory effect of peroxisome proliferator - activated receptor γ (PPARγ) agonist pioglitazone (PGZ) on the proliferation of leukemic HL-60 cells in vitro and its possible mechanism. Methods HL-60 cells were treated with different concentrations of PGZ (20-80μmol / L) for 24h, 48h and 72h. The cell growth inhibition rate was determined by MTT assay. Cell cycle and apoptosis were detected by flow cytometry (FCM) . The changes of PPARγ, Caspase-3 and PARP (ADP-ribose) polymerase (PARP) expression were detected by RT-PCR and Western blotting. Results PGZ significantly inhibited cell growth and induced cell apoptosis, showing a significant dose-effect and time-effect relationship. The result of FCM showed that the cells were mainly blocked in G0 / G1 phase, and typical sub G1 phase (apoptosis peak) appeared after treated with 60μmol / L PGZ for 48h. While PGZ induced apoptosis, PPARγmRNA and Protein expression levels are gradually increased. RT-PCR showed that the expression of Caspase-3 and PARP decreased gradually. The Western blot showed that the 32 kD Caspase-3 zymogen was activated and the 17 kD subunit fragment was activated. At the same time, the cleaved PARP of Caspase-3 89kD subunit fragment. Conclusion PGZ has a significant cell cycle arrest effect on HL-60 cells in vitro and induces cell apoptosis. It may be one of the important mechanisms by which PGZ inhibits cell proliferation and induces cell apoptosis by relying on PPARγ signaling pathway and activating Caspase-3.