干扰素-α增强足叶乙甙介导的凋亡和细胞周期停滞
Interferon-α enhances etoposide-induced apoptosis and cell cycle arrest
目的 探讨干扰素(IFN)-α对足叶乙甙介导骨肉瘤细胞周期停滞和凋亡的影响及其分子机制.方法 采用IFN-α和足叶乙甙单独或联合处理骨肉瘤U2OS细胞72 h,通过锥虫蓝法测定细胞生长抑制率,应用流式细胞术检测细胞周期停滞,通过Hochest 33258荧光染色检测凋亡,反转录-聚合酶链反应(RT-PCR)检测p53在mRNA水平的表达,通过小干扰RNA(siRNA)干扰沉默p53的表达,采用Western blot法检测p53和聚腺苷二磷酸核糖聚合酶(PARP)的表达.结果 药物处理72 h,IFN-α、足叶乙甙和联合用药组细胞生长抑制率为:(2.29±1.03)%、(23.85±3.09)%和(50.51±3.77)%,IFN-α增强足叶乙甙的生长抑制,差异有统计学意义(P=0.008).在足叶乙甙和联合用药组均出现凋亡形态学改变.足叶乙甙组和联合用药组S期细胞为(19.68±3.62)%和(11.33±2.94)%,G2/M期细胞为(65.02±2.55)%和(76.09±2.87)%,两者组间差异有统计学意义(P=0.037).IFN-α没有增加足叶乙甙引起的p53在mRNA水平的表达,但沉默p53表达后空白组、对照siRNA组、p53-siRNA组的生长抑制率分别为:(55.72±3.88)%、(51.63±4.36)%和(36.40±3.95)%,p53沉默减弱了联合用药引起的生长抑制,差异有统计学意义(P=0.023),同时联合用药组的PARP裂解明显减弱.泛半胱氨酰天冬氨酸特异性蛋白酶(Caspase)抑制剂Z-VAD-FMK预处理后Z-VAD-FMK、足叶乙甙组、IFN-α+足叶乙甙组、足叶乙甙+Z-VAD-FMK组和IFN-α+足叶乙甙+ Z-VAD-FMK组的生长抑制率为(3.52±1.98)%、(27.11±4.06)%、(56.44±4.78)%、(21.37±3.55)%、(23.81±4.22)%.Z-VAD-FMK减弱联合用药引起的生长抑制,差异有统计学意义(P =0.015),并且联合用药引起的PARP裂解也被减弱.结论 IFN-α在人骨肉瘤U20S细胞中增强足叶乙甙介导的生长抑制、细胞周期停滞和凋亡,该作用依赖p53和Caspase.
更多Objective To study effect of interferon-α (IFN-α) on etoposide-induced cell cycle arrest and apoptosis in human osteosarcoma cells with its mechanisms.Methods Osteosacoma U2OS cells were treated with IFN-α and etoposide,alone or in combination,for 72 h.Growth inhibition was determined with trypan blue exclude test.Cell cycle arrest was evaluated with FACS.Apoptosis was detected through Hoechst33258 staining.The expression of p53 was determined with reverse transcriptase-polymerase chain reaction (RT-PCR) in mRNA level.small interfering RNA (siRNA) interference was used to silence p53.Western blotting was used to evaluate the expression of p53 and poly adenosine diphosphate-ribose polymerase (PARP).Results After treatment for 72 h,the growth inhibition was (2.29 ± 1.03) %,(23.85 ± 3.09) % and (50.51 ± 3.77) % in IFN-e,Etoposide and combination group.IFN-α enhanced etoposide-induced growth inhibiton.There were statistic differences (P =0.008).There were apoptotic morphological changes in etoposide and combination groups.Cells in S phase were (19.68 ±3.62)% and (11.33 ±2.94)% in Etoposide and combination group,while G2/M cells were (65.02±2.55)% and (76.09 ±2.87)%.There were statistic differences (P=0.037).IFN-α did not further increase the expression of p53 in mRNA level.The inhibition rate was (55.72 ± 3.88) %,(51.63 ± 4.36) % and (36.40 ± 3.95) % in blank,control-siRNA and p53-siRNA group after p53 silence.There were statistic differences (P =0.023).p53 silence largely decreased PARP cleavage in response to the combination.After pan cysteinyl aspartate-specific protease (Caspase) inhibitor Z-VAD-FMK pre-treatment,the growth inhibition rate was (3.52 ± 1.98)%,(27.11 ± 4.06)%,(56.44 ± 4.78)%,(21.37±3.55)% and (23.81 ±4.22)% in Z-VAD-FMK,Etoposide,IFN-α +Etoposide,etoposide + Z-VAD-FMK and IFN-α + Etoposide + Z-VAD-FMK group.Z-VAD-FMK decreased growth inhibition caused by combination.There were statistic defferences (P=0.015).PARP cleavage caused by combination was also inhibited by Z-VAD-FMK.Conclusion IFN-α enhances etoposide-mediated growth inhibition,cell cycle arrest and apoptosis in human osteosarcoma U2OS cells.The growth inhibiton and apoptosis are p53-dependent and Caspase-dependent.
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