维A酸多种方式联合阿糖胞苷或柔红霉素对体外HL-60细胞凋亡的影响

Different ways of combination of tretinoin with cytarabine or daunorubicin lead to variable change of HL-60's apoptosis in vitro

目的探讨维A酸(ATRA)采用多种方式与阿糖胞苷(Ara-C)或柔红霉素(DNR)联用对体外白血病细胞株HL-60凋亡的影响。方法体外培养HL-60细胞株,细胞悬液以浓度2.0×105.mL-1接种后分7组,其中A组不施加任何药物,作为空白对照;B组将Ara-C与ATRA同时联合作用48 h;C组预先用ATRA作用24 h,然后改用Ara-C继续作用24 h;D组预先用Ara-C作用24 h,然后用ATRA继续作用24 h;E组将DNR与ATRA同时联合作用48 h;F组预先用ATRA作用24 h,然后用DNR继续作用24 h;G组预先用DNR作用24 h,然后用ATRA继续作用24 h。Ara-C、DNR、ATRA给药浓度均为1.0×10-7mol.L-1。应用流式细胞仪、DNA凝胶电泳及瑞氏-吉姆萨染色观察药物以不同方式联用导致细胞凋亡变化。结果D组的HL-60细胞凋亡比例轻度增加(50±14.7)%;细胞染色体完整,瑞氏染色为明显细胞凋亡。E组和G组均导致HL-60凋亡细胞明显增多,但细胞染色体丢失明显,瑞氏染色示细胞破裂。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)%。结论同时联合使用DNR与ATRA能显著减少HL-60细胞存活率。首先应用小剂量Ara-C,随后应用ATRA可较明显增多细胞凋亡数目,并能有效控制HL-60细胞死亡方式为凋亡而非坏死。

AIM To investigate variable change of apoptosis by different ways of combination of tretinoin （ATRA） with cytarabine （Ara-C） or daunorubicin （DNR） to HL-60 cell line in vitro. METHODS HL-60 cell line was cultured in vitro, eell suspension was divided into different 7 groups： group A-G,whieh respectively were： control; Ara-C and ATRA simultaneous were applied in cells for 48 h;ATRA and Ara-C subsequently were administrated respectively for 24 h including ATRA was applied first for 24 h, then followed by Ara-C for 24 h, and in verse; DNR and ATRA simultaneously were applied for 48 h ; DNR and ATRA subsequently were administrated respectively for 24 h. Drug dose of Ara-C, DNR and ATRA was 1.0 × 10^-7 mol.L^-1. The ratio of apoptosis cells was evaluated by flow eytometry. DNA gel electrophoresis was used to determine integrity of cell＇ s DNA and Wright＇ s-Giemsa＇ s staining to observe morph change of cells. RESULTS The ratio of cell apoptosis in group D increased slightly, the increase range of apoptosis was （50 ± 14.7） %. Cell＇s DNA was kept integrity and through Wright-Giemsa＇ s staining, cells appeared obvious apoptosis in morph in this group. Compared with control group, two groups including group E and G caused the apoptosis ratio significantly higher. But almost all of DNA of cells in these groups＇ was lost and Wright＇ s-Giemsa＇ s staining results showed almost all cells swelled and broke. The alive cells＇ ratio in group E can be reduced in most degree and the amount of dead cells increased to degree of （4.00 ± 0.56） %. CONCLUSION DNR in combination with ATRA can significantly decrease HL-60 survival. Low dose of Ara-C is administrated first then followed by ATRA can not only increase slightly the ratio of apoptosis of HL-60 cell, but also effectively control the ways of cell death to apoptosis not necrosis.