摘要:
目的: 研究阿糖胞苷(cytarabine,Ara-C)或柔红霉素(daunorubicine,DNR)与全反式维A酸(all-trans retinoicacid,ATRA)以不同方式联用,体外作用于白血病细胞株HL-60,导致细胞凋亡差异及可能机制。 方法: 体外培养HL-60细胞株,细胞悬液以浓度2.0×105/ml接种后分7组,分别为A:对照组;B:Ara-C与ATRA同时联用48 h组,Ara-C+ATRA48 h;C:ATRA与Ara-C先后联用48 h组,ATRA 24 h→Ara-C 24 h;D:Ara-C与ATRA先后联用48 h组,Ara-C 24 h→ATRA 24 h;E:DNR与ATRA同时联用48 h组,DNR+ATRA48 h;F:ATRA与DNR先后联用48 h组,ATRA24 h→DNR24 h;G:DNR与AT-RA先后联用48 h组,DNR 24 h→ATRA24 h。Ara-C、DNR、ATRA给药浓度均为1.0×10-7mol/L。应用流式细胞仪、Westernblotting观察药物不同方式联用致HL-60细胞凋亡的差异及相关机制变化。 结果: 与A组相比,D组HL-60细胞凋亡比例轻度增加(50±14.7)%,抗凋亡蛋白Bcl-2消失;E组和G组HL-60凋亡细胞明显增多,其中E组能最大限度减少HL-60存活数量,使HL-60细胞死亡数目增加,达(4.00±0.56)%;E、F、G组抗凋亡蛋白Bcl-2均消失。 结论: 同时联用DNR与ATRA能显著减少HL-60细胞存活率,两药的三种联用方式均可使抗凋亡蛋白Bcl-2消失。但是,首先应用小剂量Ara-C,随后应用AT-RA的联用方式可明显增多细胞凋亡数目,抑制HL-60细胞的抗凋亡蛋白Bcl-2不能表达,有效控制细胞凋亡方向,是最有潜力的药物联用方式。
Abstract:
Objective To investigate variance of apoptosis and correspondingly mechanism in HL-60 cell line in vitro by different combinations adminstration of three drug including cytarabine,daunorubicin and all-trans retinoic acid.Methonds HL-60 cell line was cultured in vitro,cell suspension was divided into different 7 groups which's density was 2.0×105/ml : Group A: control;Group B:Ara-C and ATRA were simultaneous applied in cells for 48 h(Ara-C+ATRA 48 h);Group C: ATRA and Ara-C subsequently administrated respectively for 24 h that is ATRA was applied first for 24 h,then followed by Ara-C for 24 h.Group D: Ara-C and ATRA subsequently administrated respectively for 24 h that is Ara-C was applied first for 24 h,then followed by ATRA for 24 h;Group E: DNR and ATRA simultaneously administrated for 48 h;Group F: DNR and ATRA subsequently administrated respectively for 24 h that is DNR was applied first for 24 h,then followed by ATRA for 24 h.Group G: ATRA and DNR subsequently administrated respectively for 24 h that is ATRA was applied first for 24 h,then followed by DNR for 24 h.All drug concentrations of Ara-C,DNR and ATRA were 1.0×10-7 mol/L.The ratio of apoptosis cells was evaluated by flow cytometry.Anti-apoptosis protein Bcl-2 was checked by Western Blotting. Results The ratio of cell apoptosis in D group increase slightly,the rate of increase in apoptosis was(50±14.7) %,its protein Bcl-2 disappeared.Compared with control group,both two groups including E and G groups caused the apoptosis ratio significant higher.The alive cells' ratio in E group was reduced to the highest degree and the amount of dead cells increased to a degree of(4.00±0.56) %.Anti-apoptosis protein Bcl-2 in E,F,G groups all disappeared. Conclusion Combination DNR and ATRA simultaneously can effectively decrease HL-60 survival.Three ways of combination DNR with ATRA can signifi- cantly cause protein Bcl-2 disappear.But low dose of Ara-C was administrated first then followed by ATRA can not only increase slightly the ratio of apoptosis of HL-60 cell,but also inhibited anti-apoptosis protein Bcl-2 to express and effectively controled the way of HL-60 cell's death to apoptosis,and we concluded that this combination is the most potentiall way.
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