核酶抑制端粒酶活性的实验研究  

Study on telomerase inhibition by ribozyme targeted to telomerase RNA component

作　　者：屈艺[1] 欧阳雪松[1] 刘菽秋[1] 彭晓东[1] 刘柏林[1] 

机构地区：[1]华西医科大学分子生物学研究室

出　　处：《中华医学遗传学杂志》1999年第3期133-137,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金

摘　　要：目的为有效切割肿瘤细胞端粒酶ＲＮＡ组分使端粒酶活性降低，从而使细胞不能维持端粒长度而在有限分裂后进入凋亡。方法设计并合成了针对端粒酶ＲＮＡ组分的锤头状核酶基因，构建了该核酶基因的体外转录和真核表达质粒，检测了核酶对端粒酶ＲＮＡ组分的体外切割效力和对ＨｅＬａ细胞端粒酶活性的抑制效力。结果该核酶对端粒酶ＲＮＡ组分的体外切割效率达到６０％左右。导入ＨｅＬａ细胞后使端粒酶活性降至原来的１／８～１／１０，细胞生长速度明显变慢，传至１９～２０代时出现９５％凋亡。结论该核酶可望成为有效的端粒酶抑制剂，在肿瘤基因治疗中发挥作用。Objective To evaluate the possibility of using ribozyme technology for telomerase inhibition and cancer therapy. MethodsWTBZA hammer head ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized to serve as a telomerase inhibitor. An in vitro transcription plasmid and a eukaryotic expression plasmid containing teloRZ gene were constructed. In vitro cleavage reaction was carried out by mixing the ribozyme RNA with DIGlabeledhTR in different reaction conditions. Cleavage bands were detected by digoxin chemiluminescent assay. The eukaryotic expression plasmid was inducted into HeLa cells by lipofectamine; the telomerase activities and biocharacteristics of HeLa cells were detected continuously. Results teloRZ showed a specific cleavage activity against the telomerase RNA component used as template. The in vitro cleavage ratio reached about 60%.The telomerase activities of cells expressing teloRZ dropped to eight times; the doubling times became longer and apoptosis ratios became higher with increasing population doublings (PDS); at 1920 PDS 95% cells showed apoptosis. Conclusion These findings support the potential use of this ribozyme against immortalized cancer cells.

关 键 词：核酶 端粒酶 切割 HELA细胞 肿瘤 基因治疗 

分 类 号：R73－36[医药卫生—肿瘤] R730.5[医药卫生—临床医学]