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目的:研究survivin反义寡核苷酸(ASODN)诱导慢性髓系白血病(CML)急变细胞系K562细胞凋亡及机制,并探讨与喜树碱(CPT)的活性代谢产物SN-38联合治疗的疗效。方法:Survivin ASODN转染K562细胞后,应用CCK-8法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡情况,QRT-PCR法及Western blot法检测凋亡相关基因及蛋白的变化。依据Chou-Talalay中效原理,采用calcusyn软件计算两药联合指数(CI),评价survivin ASODN与SN-38联合治疗疗效,并以Western blot检测联合治疗后凋亡相关蛋白变化。结果:Survivin ASODN呈时间和剂量依赖性抑制K562细胞增殖,24、48、72h半数抑制率(IC50)分别约为600、498、296nmol/L。600nmol/L survivinASODN以时间依赖方式诱导K562细胞凋亡,下调survivin mRNA及蛋白表达,而Bcl-2蛋白表达无明显变化。survivin ASODN与SN-38联合治疗K562细胞48h,两药极低浓度时为拮抗及相加作用(Fa≤0.12),随浓度增大表现剂量依赖性的协同作用(Fa>0.12),两药最强协同效应时浓度为380nmol/L survivin ASODN和38nmol/L SN-38。联合组的survivin及Bcl-2蛋白表达较2单药组及对照组均明显下降。结论:survivin ASODN抑制K562细胞生长并诱导凋亡,和SN-38联合治疗通过下调survivin及Bcl-2表达发挥协同效应(Fa>0.12)。
OBJECTIVE: To investigate the apoptosis of K562 cells induced by survivin antisense oligonucleotide (ASODN) and to explore the possible mechanism of the combination therapy with SN-38, the active metabolite of CPT Efficacy. Methods: After transfecting K562 cells with Survivin ASODN, the cell proliferation inhibition rate was detected by CCK-8 assay and the apoptosis was detected by flow cytometry. The changes of apoptosis related genes and proteins were detected by QRT-PCR and Western blot. According to the principle of Chou-Talalay, calcusyn software was used to calculate the combined effect of two drugs (CI) to evaluate the therapeutic effect of survivin ASODN combined with SN-38. Western blot was used to detect the changes of apoptosis related proteins. RESULTS: Survivin ASODN inhibited the proliferation of K562 cells in a dose-and time-dependent manner. The IC50 of 24,48,72 h were about 600,498 and 296 nmol / L, respectively. The apoptosis of K562 cells was induced by 600 nmol / L survivin ASODN in a time-dependent manner, and the expression of survivin mRNA and protein was down-regulated while the expression of Bcl-2 protein did not change significantly. K562 cells were treated with survivin ASODN in combination with SN-38 for 48h. Antagonist and additive effects were observed at very low concentration (Fa≤0.12) and dose-dependent synergistic effect (Fa> 0.12) Strong synergistic effect when the concentration of 380nmol / L survivin ASODN and 38nmol / L SN-38. The combined group of survivin and Bcl-2 protein expression than 2 single drug group and control group were significantly decreased. CONCLUSIONS: Survivin ASODN can inhibit the growth of K562 cells and induce apoptosis. Synergistic effect of survivin ASODN and SN-38 treatment is shown by down-regulation of survivin and Bcl-2 expression (Fa> 0.12).