反义hTERT基因对白血病细胞端粒酶表达及活性的抑制作用  

Antisense hTERT inhibits gene expression and functional activity of telomerase in leukemia cell lines

作　　者：孙来保[1] 李成荣[1] 文剑明[2] 王国兵[1] 张萌[2] 杨军[1] 李若馨[1] 

机构地区：[1]深圳市儿童医院病理科,518026 [2]中山大学中山医学院病理教研室

出　　处：《中华病理学杂志》2004年第5期454-457,共4页Chinese Journal of Pathology

基　　金：深圳市卫生科技计划基金资助项目 ( 19980 5 0 2 1)

摘　　要：目的　探讨反义hTERT基因体外对白血病细胞端粒酶基因表达及其活性的抑制作用。方法　采用基因重组技术设计并构建了针对端粒酶活性蛋白催化亚单位mRNA的 5′端序列的反义pcDNA hTERT真核表达载体 ,通过SuperFect(QIAGEN)将其瞬时转染人早幼粒白血病细胞株HL60 及人红白血病细胞株K562 ,并运用流式细胞术测定其细胞凋亡与细胞周期的变化 ,同时运用实时荧光定量逆转录 聚合酶链反应方法对其端粒酶蛋白催化亚单位基因的表达水平进行了检测 ,端粒酶活性的检测则采用了非放射性TRAP 银染法。结果　成功地构建了反义pcDNA hTERT真核表达载体 ,并将其转染至K562 和HL60 白血病细胞株。结果与空载体组、空白组相比 ,反义 pcDNA hTERT组体外显示出明显有效地抑制端粒酶的基因表达及其活性的作用 ;在细胞凋亡与细胞周期方面 ,反义组能引起细胞周期左移 ,增殖分裂能力降低 ,同时伴有细胞凋亡数的增加。结论　研究构建的反义hTERT基因体外确实能明显有效地下调白血病细胞端粒酶的基因表达及其活性 。Objective To explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment.Methods An antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5′ end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL_(60) and K_(562)) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively. Results An antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control). Conclusion In-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.

关 键 词：反义HTERT 白血病细胞 体外 端粒酶 细胞周期 细胞凋亡 酶表达 DNA 基因 真核表达载体 

分 类 号：R733.7[医药卫生—肿瘤] R734.2[医药卫生—临床医学]