端粒酶反义寡核苷酸治疗脑胶质瘤实验研究

目的研究端粒酶反义寡核苷酸对胶质瘤细胞端粒酶活性、细胞增殖和细胞凋亡的影响,为以端粒酶为靶点治疗脑胶质瘤提供理论和实验依据。方法以端粒酶催化亚单位基因(hTERT)为靶点设计合成硫代磷酸化反义寡核苷酸(PS-ASODN),经脂质体(lipofectin)包裹转染胶质瘤细胞,每隔24h取样。采用甲基噻唑基四唑(MTT)法检测PS-ASODN对细胞生长增殖的影响;用端粒重复序列扩增-聚合酶链反应-酶联免疫吸附测定(TRAP-PCR-ELISA)法检测端粒酶活性的变化;用流式细胞仪检测细胞周期和凋亡的改变。结果PS-ASODN实验组与正义组、错义组及空白对照组相比较,48h后细胞生长增殖受抑制,端粒酶活性降低(P<0.05),72h时,差异有高度显著性(P<0.01);实验组对细胞凋亡有显著的促进作用,细胞多阻滞于G2/M期。其作用具有序列选择的特异性,在一定的时间范围内,呈时间依赖性。结论端粒酶反义寡核苷酸能够有效地抑制胶质瘤细胞的生长而促进其凋亡,具有较好的临床应用前景。

The experimental study of telomerase antisense oligonucleotide in the treatment of glioma cells

Objective To study the curative effect of telomerase antisence oligonucleotides on telomerase activity and proliferation of glioma cells, and to provide further insight on telomerase as a target in the treatment of malignant glioma.Methods After encapsulated with lipofectin, the phosphorothioate antisensce oligonucleotide (PS-ASODN) of telomerase was co-incubated with cultured glioma cells. With methyl thiazolyl tetrazolium (MTT) method , the cell survival rate was detected. Telomeric repeat amplification protocol-polymerase chain reaction- enzyme -linked immunoadsordent assay (TRAP-PCR-ELISA) was used to examine the telomerase activity. Cell apoptotic rate and the phase of cell cycle were determined by flow cytometry.Results Compared with PS-sense ODN and PS-missense ODN, PS-ASODN inhibited cell proliferation and telomerase activity after being applied for 48 h (P<0.05), and demonstrated significant difference at 72 h (P<0.01). After 96 h, the growth of most transfected glioma cells stopped at G_ 2 /M phase, and the number of apoptosis cells increased as time went on. The efficacy had the sequence selectivity and depended on time in a certain range.Conclusion PS-ASODN inhibits glioma cells proliferation and their telomerase activity, so it plays a potential role in glioma treatment.