人端粒酶逆转录酶核酶抑制端粒酶活性  

Inhibition of Telomerase Activity by Ribozyme Targeted to Human Telomerase Transcriptase

作　　者：屈艺[1] 刘菽秋[1] 彭文珍[1] 刘柏林[1] 

机构地区：[1]四川大学华西基础医学院分子生物学开放实验室,成都610041

出　　处：《生物化学与生物物理学报》2002年第3期323-328,共6页

基　　金：国家自然科学基金资助项目 (No .39970 836 )~~

摘　　要：为有效切割端粒酶逆转录酶mRNA以降低端粒酶活性 ,从而使肿瘤细胞生长变慢 ,凋亡增加。设计并合成了针对端粒酶逆转录酶mRNA的锤头状核酶基因 ,构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中 ,检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明 ,该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA ;在细胞内能明显抑制端粒酶活性 ,使细胞生长变慢 ,倍增时间延长。因而 ,该核酶可望成为有效的端粒酶抑制剂 。Human telomerase reverse transcriptase (hTERT) is the catalytic subunit and the key factor which controls the telomerase activity, so it is the best choice to inhibit telomerase through controling hTERT expression. In this work, a hammer head ribozyme directed against the hTERT mRNA (hTERTRZ) was designed and synthesized to serve as a telomerase inhibitor. In order to test its in vitro cleavage activity, two in vitro transcription plasmids containing hTERTRZ and hTERT gene respectively were constructed. Ribozyme RNA and DIG-labeled-hTERT were synthesized by in vitro transcription. In vitro cleavage reactions were carried out by mixing the hTERTRZ with DIG-labeled-hTERT under different reaction conditions, and cleavage bands were detected by digoxin chemiluminescent assay. hTERTRZ showed a specific cleavage activity against the hTERT used as template. To investigate its in vivo effect of telomerase inhibition in tumor cells, a eukaryotic expression plasmid containing the hTERT ribozyme gene was introduced into HeLa cells and hepatoma cells by using LipofectAMINE. In the transfectants, the level of intact hTERT mRNA and the telomerase activity were clearly reduced, and the telomere length of these clones was apparently shortened at the beginning period, then kept a fixed value without further shortening. All the transfectants with ribozyme grew clearly more slowly than the parental cell line. The doubling time of the tansfectants prolonged compared to the negative control, but no apparent apoptosis was shown even at their 37th passage. These findings suggest the potential application of this ribozyme as a new theraputic agent directed against immortalized cancer cells.

关 键 词：人端粒酶 逆转录酶 核酶 抑制 端粒酶活性 肿瘤发生 

分 类 号：Q55[生物学—生物化学] R73－3[医药卫生—肿瘤]