hTERT反义寡核苷酸增强DDP介导细胞凋亡作用  

hTERT antisense oligonucleotides enhance apoptosis induced by cisplatin in HeLa cell lines

作　　者：周波[1] 陈忠东[1] 

机构地区：[1]南华大学第一附属医院妇产科,湖南衡阳421001

出　　处：《中国药理学通报》2007年第12期1645-1650,共6页Chinese Pharmacological Bulletin

摘　　要：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。Aim To investigate the effects of human telomerase reverse transcriptase （hTERT） antisense phosphorothioate oligodeoxynucleotide（ASODN） on telomerase activity and apoptosis induced by crisplatin （DDP） in HeLa cell lines. Methods HeLa cells were collected after transfection of 0. 05, 0. 1,0. 2 μmol·L^-1 of ASODN and the total DNA was extracted. Telomeric repeat amplification protocol （ TRAP ） -AgNO3 stain was used to detect the activity of telomerase. Apoptosis of HeLa cells was detected by AO staining morphological observation and flow cytometry（FCM） analysis technology. Results The telomerase activity of HeLa cells was expressed in high level, which was significantly reduced dose-dependently and time-dependently after treatment with ASODN of hTERT. Two days after transfeetion of ASODN of 0. 05 μmol·L^-1, 0. 1μmol·L^-1, 0. 2 μmol·L^-1, the telomerase activity decreased by 21.77% ,52. 42% and 71.10% respectively. After treatment with ASODN for 24,48 h and 72 h, the telomerase activity decreased by 12. 48% ,38.27% and 71.10% respectively. In morphological observation using acridine orange （AO）staining method, HeLa cells displayed typical apoptotic figure after treated with DDP plus ASODN of hTERT. The percentage of apoptosis of HeLa cells treated with DDP of 1.5 mg·L^-1, 3.0 mg·L^-1, for 48 hours after 24 hours exposure to ASODN of 0. 2 μmol·L^-1 （50. 35%, 29. 67% ） was significantly higher than that treated with DDP alone （19.67%, 11.38%, P 〈 0.05）, or combination of DDP and RSODN of hTERT（ 19. 33%, 12. 13% ,P 〈 0. 05）. Condusions Human telomerase reverse transcriptase （hTERT） antisense phosphorothioate oligode- oxynucleotide（ASODN） possessed inhibitory effects of telomerase activity in HeLa cells line and could enhance the DDP-induced apoptosis of HeLa cells.

关 键 词：HTERT 反义核酸 端粒酶 凋亡 顺铂 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R342.4[医药卫生—基础医学]