摘要
目的 探讨反义封闭胶质瘤细胞端粒酶逆转录酶（hTERT）基因表达,对其端粒长度及p33ING1b表达的影响.方法 用真核表达质粒（pcDNA3.0）构建hTERT正义和反义RNA表达载体.经脂质体介导,将空载体peDNA3.0及正义和反义RNA表达载体分别导人人胶质母细胞瘤细胞株（TJ905细胞）,经G418筛选获得分别携带空载体、hTERT正义或反义RNA表达载体的三种TJ905细胞亚克隆.用Western blot、Tolo TAGGG端粒长度分析和RT-PCR方法,检测各对照组和反义封闭组不同代数TJ905细胞的hTERT蛋白表达、端粒长度及p33ING1b mRNA转录.结果 空白对照组、空载体组及正义RNA组TJ905细胞均有hTERT蛋白异常表达、端粒缩短迟缓及p33ING1b mRNA表达异常减少,以上三个对照组间这些指标的差异均无统计学意义（P〉0.05）.与以上三组相比,反义封闭组TJ905细胞的hTERT蛋白表达水平明显降低（P〈0.01）,端粒长度明显缩短（P〈0.01）,而p33ING1b mRNA表达水平则明显升高（P〈0.01）,它们各自与三个对照组相同指标之间的差异均有统计学意义.结论 胶质瘤细胞hTERT过表达导致的端粒酶异常再活化,可通过维持有效端粒长度抑制其抑癌因子p33ING1b表达,反义封闭hTERT基因表达可矫正该异常过程,提示hTERT是胶质瘤分子靶向治疗的重要靶点.
Objective To investigate the effect of antisense RNA of human telomerase reverse transcriptase（ hTERT） on telomere length and p33ING1b expression in glioma cells. Method Recombinant plasmids expressing sense RNA[pcDNA - hTERT （ ＋ ）] and antisense RNA[pcDNA - hTERT （ - ）] of hTERT were constructed with eukaryotic expression plasmid pcDNA3.0. The empty plasmid, pcDNA -hTERT（ ＋ ） or pcDNA - hTERT（ - ） were transfered into the human glioblastoma cell line（TJ905 cells） with liposome respectively. Three subclones of TJ905 cells loading empty plasmid, pcDNA - hTERT （ ＋ ） or pcDNA -hTERT（ - ） respectively were established by G418 selection. Their expressions of hTERT protein and p33ING1b mRNA were detected by Western Blot and RT - PCR. The telomere length was analysed using Tolo TAGGG Telomere Length Assay reagent kit Results All of the hTERT protein level, telomere length and p33ING1b mRNA level had not significant difference among TJ905 cell control,the subclones of TJ905 cell with empty plasmid or pcDNA - hTERT （ ＋ ） （ P 〉 0. 05 ） . As compared with above three groups, the expression of hTERT protein decreased （ P 〈 0. 01）, the telomere length shortened （ P 〈 0. 01） and the expression of p33ING1b mRNA was up - regulated（ P 〈0. 01） significantly in the subclone of TJ905 cell with pcDNA - hTERT （ - ） . Conclusions The abnormal reactivation of telomerease resulted fromoverexpression of hTERT may inhibit expression of suppressor, p33ING1b ,by maintaining the effective telomere length in the glioma cells.Antisense blocking expression of hTERT gene is able to rectify the abnormal process.Above results suggest that hTERT is the important target for the glioma molecular target-therapy.
出处
《中华神经外科杂志》
CSCD
北大核心
2010年第7期652-656,共5页
Chinese Journal of Neurosurgery
基金
基金项目：国家“973计划”分项目（2010CB529405）
国家自然科学基金资助项目（30770827）
天津市高等学校科技发展基金重点项目（2004ZD06）
教育部高等学校博士学科点专项科研基金（20060062005）
天津市科技支撑计划重点项目（07ZCKFSF00800）
天津市高等学校科技发展基金计划项目（20060202）
