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目的探讨Z-ajoene诱导细胞周期阻断于G2/M期的分子机制以及对端粒酶活性的抑制作用。方法采用MTT法测定Z-ajoene对HL-60细胞的增殖抑制率;以流式细胞仪测定Z-ajoene对HL-60细胞的周期阻断作用,并采用Westernblot法测定相关蛋白p34cdc2、cyclinB1、cyclinA的表达水平;TRAP-银染法测定端粒酶活性的变化,同时采用RT-PCR方法检测端粒酶hTRT和TP1mRNA的表达。结果10μmol/LZ-ajoene作用后能明显将HL-60细胞阻滞于G2/M期。在药物作用10h后,G2/M期细胞的比例达到顶峰,与对照组比较增加了1.95倍。另外,10μmol/LZ-ajoene作用后出现cyclinB1蛋白的聚集及p34cdc2蛋白表达的降低,但cyclinA蛋白的水平并无显著性变化。经Z-ajoene作用24h后,HL-60细胞端粒酶活性显著降低。而不同浓度的药物作用12h,其端粒酶活性均无显著性变化。在10μmol/LZ-ajoene作用24h后,能够降低端粒酶hTRT和TP1mRNA的水平。结论Z-ajoene能够在作用较短时间内明显将细胞周期阻断于G2/M期,同时伴有cyclinB1聚集及p34cdc2的抑制;而HL-60细胞的端粒酶活性仅在药物作用较长时间后才有一定降低,并且端粒酶hTRT和TP1mRNA的表达也同时降低。结果表明,Z-ajoene的细胞周期阻断作用可能是造成端粒酶缺失及HL-60细胞生长抑制的重要原因。
Objective To investigate the molecular mechanism of Z-ajoene-induced G2 / M arrest in cell cycle and its inhibitory effect on telomerase activity. Methods The inhibitory rate of Z-ajoene on the proliferation of HL-60 cells was determined by MTT assay. The cell cycle arrest of HL-60 cells by Z-ajoene was determined by flow cytometry. The expressions of p34cdc2, cyclinB1, cyclinA The telomerase activity was detected by TRAP-silver staining. Meanwhile, the expression of telomerase hTRT and TP1 mRNA was detected by RT-PCR. Results After HL-60 cells were treated with 10μmol / L Z-ajoene, HL-60 cells were arrested in G2 / M phase. After 10 hours of drug treatment, the proportion of cells in G2 / M phase reached the peak, an increase of 1.95 times compared with the control group. In addition, cyclinB1 protein accumulation and p34cdc2 protein expression decreased after treated with 10μmol / L Z-ajoene, but the level of cyclinA protein did not change significantly. After treated with Z-ajoene for 24 h, the telomerase activity of HL-60 cells was significantly decreased. However, no significant change of telomerase activity was observed after different concentrations of drug were applied for 12h. After treated with 10μmol / L Z-ajoene for 24h, telomerase hTRT and TP1mRNA could be decreased. Conclusion Z-ajoene can significantly block the cell cycle in G2 / M phase in a short period of time, accompanied by cyclinB1 aggregation and p34cdc2 inhibition; and HL-60 cell telomerase activity only in the role of drug for a long time After a certain decrease, and the expression of telomerase hTRT and TP1 mRNA are also reduced. The results showed that the cell cycle arrest of Z-ajoene may be an important reason for the loss of telomerase and the inhibition of the growth of HL-60 cells.