全硫代反义寡核苷酸对脑胶质瘤细胞端粒酶活性作用

Inhibitory effects of the proliferation of glioma cells by antisense phosphorothioate oligonucleotide

目的本研究采用针对人端粒酶hTR基因的反义寡聚脱氧核苷酸,探讨端粒酶反义寡聚脱氧核苷酸(antisense oligodeoxy nucleotides,ASODN)对脑胶质瘤HO-8910细胞端粒酶活性及细胞增殖的影响。方法本实验将互补于hTR模板区的全硫代修饰型反义寡核苷酸(PS-ASODN)经lipotap脂质体转染作用于脑胶质瘤细胞系C6,然后分别采用四甲基偶氮唑蓝(MTT)比色实验、酶联免疫吸附试验(ELISA)法和流式细胞术检测C6脑胶质瘤细胞的体外增殖、端粒酶活性、细胞凋亡和细胞周期的改变。结果经MTT法检测,PS-ASODN对C6细胞生长具有明显的抑制作用和诱导细胞凋亡,1.0μMPS-ASODN处理后24h细胞生长抑制率为12.93%,且随着时间的延长,抑制作用逐渐增强;给药72h对细胞生长抑制率达到最高,为50.81%,有相应的浓度依赖关系。ELISA法检测结果 ,0.5～1.5μM的PS-ASODN对C6细胞作用72h后端粒酶皆为阴性,表明PS-ASODN能够抑制端粒酶活性,对照组端粒酶皆为阳性。结论端粒酶PS-ASODN对体外培养的脑胶质瘤C6细胞的增殖有明显的浓度、时间依赖性抑制作用;抑制脑胶质瘤C6的端粒酶活性和诱导C6脑胶质瘤细胞发生凋亡,可能是端粒酶PS-ASODN抑制C6细胞增殖的主要机制。

Objective To study the effect of telomerase phosphorathioate antisense oligodeoxy nucleotides （PS-ASODN） on telomerase activity and proliferation of C6 glioma cells. Methods The anti-proliferation effect of PS-ASODN on C6 glioma cells was determined by MTT assay. The activity of telomerase was tested by ELSIA, apoptosis and cell cycle were examined by flow cytometer method. Results The growth and apoptosis of C6 glioma cells were inhibited and induced respectively by 0.5～1.5μM telomerase PS-ASODN, the inhibitory rate of 1.0μM PS-ASODN was 12.93% 24 h after administration of PS-ASODN,reached 50.81% 72 h after administration of PS-ASODN （ P〈0.01）. The activity of C6 glioma cells telomerase was repressed by 0.5～1.5μM PS-ASODN 72 h after administration of PS-ASODN, with earlier spike when the C6 glioma cells were treated with 1.5μM PS-ASODN at 72 h. C6 glioma cells treated with 1.5μM PS-ASODN were arrested in Gl/G0. Conclusions PS-ASODN had significant inhibitory effects on the proliferation of the C6 glioma cell with time-dependent and dose dependent manners, whose mechanism might be repated to the telomerase enzyme activity and induction of C6 glioma cell apoptosis