摘要目的 探讨叠氮胸苷(AZT)对TJ905 人胶质母细胞瘤细胞株端粒酶活性、增殖和凋亡的影响及其机制.方法 体外培养TJ905细胞分别经50、100及200 μmol/L叠氮胸苷处理24 h后,用端粒重复扩增法检测端粒酶活性及集落形成效率;继续培养并维持相应叠氮胸苷浓度,取第1、3和6代细胞,利用Western blot检测cyclin A表达水平,用流式细胞术检测细胞周期分布并结合单细胞凝胶电泳检测细胞凋亡,用Ki-67免疫细胞化学染色检测细胞增殖活性.结果 叠氮胸苷可抑制TJ905细胞的端粒酶活性.50和100 μmol/L组cyclin A表达水平均显著低于对照组(P<0.01),并均呈时间和剂量依赖性降低.三个用药组的Go/G1期细胞均显著少于对照组(P<0.05～0.01),S期细胞均显著多于对照组(P<0.05～0.01);在各用药组第1代GO/G1期细胞减少和S期细胞增加均呈剂量依赖性;各用药组S期细胞增加均呈显著的时间依赖性,而Go/G1期细胞减少仅在50 μmol/L组呈时间依赖性.各用药组第1和6代的凋亡细胞数均显著多于对照组(P<0.05～0.01);各用药组第6代的凋亡细胞数随用药浓度升高而相应增加(P<0.05～0.01),且均多于同浓度组的第1和第3代(P<0.05～0.01).各用药组集落形成效率及Ki-67阳性细胞数均显著低于对照组(P<0.01),二者还随用药浓度增加和(或)作用时间延长相应减少.对照组各代间以上各指标的差异均无统计学意义(P>0.05).结论 叠氮胸苷可通过抑制端粒酶活性抑制TJ905细胞cyclin A表达,阻止该细胞从S期向G2期过渡,发挥其抑制增殖和诱导凋亡的作用.

abstractsObjective To investigate the pharmacological effects and underlying mechanism of azidothymidine (AZT) on human glioblastoma cells in vitro. Methods The telomerase activity of human glioblastoma TJ905 cells was determined by TRAP assay after 24 hrs' incubation with 50, 100,200 μmol/L AZT and control vehicle solution. Colony formation efficiencies of the cells were recorded. Cells of the 1 st,3rd and 6th generations were harvested, followed by evaluations of cyclin A protein expression by Western blot, cell cycle distribution by flow cytometry, apoptotic level by single cell gel electrophoresis and proliferation index by Ki-67 immunocytochemical staining. Results AZT inhibited telomerase activity of TJ905 cells. Cyclin A expression levels in the cells treated with 50 and 100 μmol/L AZT were significantly lower than controls (P<0.01), and down-regulation of the expression was in a dose- and time-dependent manner. Compared with controls, Go/G1 phase cells were obviously decreased ( P<0.05～0.01)and S phase cells significantly increased (P<0.05～0.01) after treatment with 50, 100 and 200 μmol/L AZT. The cell numbers of Go/G1 and S phases at the 1 st generation of above three treated groups changed in a dose-dependent manner, whereas S phase cells increases in all AZT treatment groups and Go/G1 phase cell decrease in group treated with 50 μmol/L AZT were also in a time-dependent manner. Both the apoptotic cells of the 1st and 6th generations of all AZT treatment groups were significantly more than controls (P<0.05～0.01), their numbers of the 6th generations of the three groups increased with AZT concentration (P<0.05～0.01), and all of them were more than the 1st and 3rd generations of the same dosage group (P<0.05～0.01). Colony formation efficiencies and Ki-67 labeling indexes of the three AZT treatment groups were distinctly lower than controls (P<0.01), and they were also decreased with the elevation of AZT concentration and/or the elongation of the incubating time. The difference of any above parameter had no significance among the 1st, 3rd and 6th generations of control group (P>0.05).Conclusion AZT blocks S/G2 conversion of TJ905 cells by inhibition of telomerase activity and cyclin A expression, leading to an enhancement of apoptesis and suppression of cell proliferation.