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目的:研究拓扑替康(TPT)对骨髓增生异常综合征(MDS)急性髓系白血病(AML)M2变细胞系MUTZ-1的作用机制,以及环磷酰胺(CTX)对TPT作用靶标Topo-ⅠmRNA表达水平的影响。方法:MTT法测定TPT对MUTZ-1细胞的杀伤作用。流式细胞术分析TPT对MUTZ-1细胞周期的影响。染色体分析方法观察TPT对MUTZ-1的影响。半定量RT-PCR检测CTX对Topo-ⅠmRNA表达水平的影响。结果:①5、10、20、40、80、160nmol/L TPT与MUTZ-1细胞共培养72h后细胞存活率呈剂量依赖性性下降,分别为(78.9±10.7)%、(69.2±4.7)%、(58.6±7.1)%、(49.9±7.6)%、(42.4±0.8)%和(23.7±9.8)%,各实验组与对照组(505.6±20.1)%之间均差异有统计学意义(均P<0.05)。②TPT阻碍MUTZ-1细胞染色体发生分离,并使MUTZ-1细胞发生G2/M期阻滞,该阻滞呈时间依赖性和剂量依赖性。③半定量RT-PCR发现0.224mmol/LCTX与MUTZ-1细胞共培养24、48和72h后,Topo-ⅠmRNA表达都上升,以48h的表达量最高。结论:TPT对MUTZ-1细胞的生长抑制呈剂量依赖性。染色体分离受阻和G2/M期阻滞是TPT的作用机制之一。CTX可上调Topo-Ⅰ表达,与TPT发挥联合抗白血病作用。
OBJECTIVE: To investigate the mechanism of topotecan (TPT) on MUTZ-1 in M2 cell line of myelodysplastic syndrome (MDS) acute myeloid leukemia (AML) and to investigate the effects of cyclophosphamide (CTX) on TPT target Topo-I mRNA Effect of expression level. Methods: MTT assay was used to determine the killing effect of TPT on MUTZ-1 cells. The effect of TPT on the cell cycle of MUTZ-1 cells was analyzed by flow cytometry. Chromosome analysis was used to observe the effect of TPT on MUTZ-1. Semi-quantitative RT-PCR was used to detect the effect of CTX on Topo-Ⅰ mRNA expression. RESULTS: ① The viability of cells treated with 5, 10, 20, 40, 160, 160 nmol / L TPT for 72 hours was (78.9 ± 10.7)%, (69.2 ± 4.7)% , (58.6 ± 7.1)%, (49.9 ± 7.6)%, (42.4 ± 0.8)% and (23.7 ± 9.8)%, respectively. The differences between the experimental and control groups (505.6 ± 20.1)% were statistically significant All P <0.05). (2) TPT blocked chromosomes of MUTZ-1 cells and arrested G2 / M phase in MUTZ-1 cells in a time-and dose-dependent manner. ③ Semi-quantitative RT-PCR showed that Topo-ⅠmRNA expression increased after 0.224mmol / L LCTX and MUTZ-1 cells were co-cultured for 24,48 and 72h, respectively. Conclusion: TPT inhibits the growth of MUTZ-1 cells in a dose-dependent manner. Obstruction of chromosome segregation and G2 / M phase arrest is one of the mechanisms of TPT. CTX can up-regulate Topo-Ⅰ expression and play a joint anti-leukemia effect with TPT.