阿糖胞苷或柔红霉素与全反式维A酸的不同联用方式致体外HL-60凋亡差异

Different administrations of combination cytarabine or daunorubicin with all-trans retinoic acid lead to variance of HL-60''s apoptosis and respectively mechanism in vitro

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。

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 retinoie acid. nethonds HL-60 cell line was cultured in vitro, cell suspension was divided into different 7 groups which＇ s density was 2.0 × 10 5/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 b, then followed by Ara-C for 24 b. 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 ceils 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 significantly 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 eontroled the way of HL-60 cell＇s death to apoptosis, and we concluded that this combination is the most potentiall way.