端粒酶逆转录酶核酶hTERT-5′RZ抑制HeLa细胞端粒酶活性的实验研究  

Attenuation of telomerase activity by hammerhead ribozyme targeting the 5′-end of hTERT mRNA

作　　者：屈艺[1] 刘菽秋[1] 刘柏林[1] 

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

出　　处：《中华医学遗传学杂志》2002年第5期389-392,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金 (39970 836)~~

摘　　要：目的　有效抑制肿瘤细胞端粒酶活性 ,为肿瘤的基因治疗提供依据。方法　设计合成了针对端粒酶逆转录酶 m RNA5′端区的锤头状核酶 (5′- end of human telomerase reverse trascriptase m RNA,h TERT- 5′RZ)基因 ,并将该基因重组入 pc DNA3.1(+) ,经体外转录得到小片段 h TERT- 5′RZ。用脂质体介导法 ,将 h TERT- 5′RZ导入 He L a细胞以检测其对 He L a细胞端粒酶活性的抑制效力。同时比较了同法合成的针对端粒酶 RNA模板的核酶 (telomerase m RNA,telo RZ)和两种核酶混合物对 He L a细胞端粒酶活性的抑制效力。结果　小片段 h TERT- 5′RZ能有效抑制 He L a细胞端粒酶活性 ,且其抑制效力较单纯telo RZ为高 ,两种核酶混合物的端粒酶抑制效力与 h TERT- 5′RZ相当 ,也高于单纯 telo RZ。结论　小片段核酶 h TERT- 5′RZ可望成为优于 telo RZ的端粒酶抑制剂 ,在肿瘤基因治疗中发挥作用。Objective: To assess the telomerase activity inhibitory effect of hammerhead ribozyme targeting the 5′-end of human telomerase reverse transcriptase mRNA (hTERT-5′RZ), to compare it with the effect of another ribozyme telomerase mRNA (teloRZ), and the combine the applications of the two ribozymes. Methods: hTERT-5′RZ gene was synthesized and cloned into pcDNA3. 1(+); the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of pSPT19-teloRZ which had been constructed by the present authors. The ribozymes were transiently transfected into HeLa cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed. Results: The ribozyme targeting the 5′-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combined use of hTERT-5′RZ and teloRZ also showed clear inhibitory activity, but the inhibitory effect of teloRZ used alone was not so strong. Conclusion: These observations suggest that the use of hTERT-5′RZ and the combined use of hTERT-5′RZ and teloRZ are more effective in telomerase inhibition as compare with the use of teloRZ alone. They may find applications in cancer therapy.

关 键 词：端粒酶 端粒酶逆转录酶 HELA细胞 核酶 基因治疗 肿瘤治疗 

分 类 号：R346[医药卫生—基础医学]