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目的研究端粒酶催化亚基(hTERT)反义寡核苷酸(ASODN)对HeLa细胞端粒酶活性的抑制及其对顺铂(DDP)诱导细胞凋亡的影响。方法用逆转录聚合酶链反应技术(RT-PCR)定量端粒酶重复扩增法(TRAP)检测细胞的端粒酶活性。观察细胞凋亡的形态学变化,流式细胞仪对细胞凋亡进行定量分析。结果ASODN作用后,细胞的端粒酶活性明显降低,且这种作用具有明显的时间和剂量依赖性。0.05、0.1μmol.L-1和0.2μmol.L-1的硫代ASODN治疗后,HeLa细胞的端粒酶活性分别下降了21.8%、52.4%和71.1%。0.2μmol.L-1的硫代ASODN作用HeLa24、48h和72h后,细胞的端粒酶活性分别下降了12.48%、38.27%和71.10%。细胞转染0.2μmol.L-1浓度的ASODN24h后再与1.5、3.0mg.L-1浓度的顺铂联合作用,吖啶橙染色可见典型的凋亡形态,并且凋亡百分率(50.35%、29.67%)分别与RSODN联合顺铂组(19.33%、12.13%)、单用顺铂组(19.67%、11.38%)比较,差异有显著性(P<0.05)。结论hTERT反义寡核苷酸能有效抑制其端粒酶活性,并且促进DDP诱导HeLa细胞凋亡。
Objective To study the effect of hTERT antisense oligodeoxynucleotide (ASODN) on telomerase activity in HeLa cells and its effect on apoptosis induced by cisplatin (DDP). Methods Telomerase activity was detected by reverse transcription-polymerase chain reaction (RT-PCR) and telomerase repeat amplification (TRAP). Morphological changes of apoptosis were observed, and apoptosis was quantitatively analyzed by flow cytometry. Results ASODN effect, the cell telomerase activity was significantly reduced, and this effect has a significant time and dose-dependent. The telomerase activity of HeLa cells decreased by 21.8%, 52.4% and 71.1% respectively when treated with 0.05, 0.1μmol.L-1 and 0.2μmol.L-1 ASODN. After treated with 0.2 μmol·L-1 ASODN for 24 h, the telomerase activity decreased by 12.48%, 38.27% and 71.10%, respectively. Cells were transfected with 0.2μmol.L-1 ASODN for 24h and then treated with 1.5, 3.0mg.L-1 cisplatin. Apoptotic morphology was observed by acridine orange staining, and the percentage of apoptosis was 50.35%, 29.67 % Respectively) was significantly higher than that of RSODN combined with cisplatin (19.33%, 12.13%) and cisplatin alone (19.67%, 11.38%), respectively (P <0.05). Conclusion hTERT antisense oligonucleotide can effectively inhibit its telomerase activity, and promote DDP-induced HeLa cell apoptosis.