中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (11): 685-688.

• 论著 • 上一篇    下一篇

hTERT-siRNA及hTR-siRNA的合成及对Hut78细胞端粒酶活性的影响

徐秀莲1, 戚金亮2, 陈声利1, 姜祎群1, 曾学思1, 孙建方1   

  1. 1. 中国医学科学院、中国协和医科大学皮肤病医院病理科, 南京 210042;
    2. 南京大学生命科学学院
  • 收稿日期:2005-02-25 发布日期:2005-11-15
  • 通讯作者: 孙建方, E-mail:sunjf@hotmail.com E-mail:sunjf@hotmail.com
  • 基金资助:
    国家教委高校博士点基金资助项日(20030023055)

Synthesis of hTERT-siRNA and hTR-siRNA by in vitro Transcription and Their Inhibition on Telomerase Activity in Hut78 Cells

XU Xiu-lian1, QI Jin-liang2, CHEN Sheng-li1, JIANG Yi-qun1, ZENG Xue-si1, SUN Jian-fang1   

  1. Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, China
  • Received:2005-02-25 Published:2005-11-15

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摘要： 目的 探讨合成端粒酶逆转录酶(hTERT)基因及端粒酶RNA(hTR)的小干扰RNA(small interfering RNA,siRNA)对皮肤T细胞淋巴瘤(CTCL)细胞株Hut78端粒酶活性的影响.方法 采用体外转录法分别合成两条hTERT—siRNA及hTR—siRNA.采用siRNA直接处理细胞裂解液及磷酸钙共沉淀法将siRNA转染入Hut78细胞两种处理方式,应用端粒酶重复序列扩增聚合酶链反应(TRAP—PCR)及聚丙烯酰胺凝胶电泳银染检测端粒酶活性的变化.结果 体外转录法能高效合成siRNA,产量达22.4μg/40μLsiRNA合成体系.经250ngsiRNA直接处理细胞裂解液后,可高效降低端粒酶的活性,抑制率达87%左右.30ng siRNA转染细胞36h后,可降低端粒酶活性20%左右,而250ngsiRNA可降低75%左右.结论 体外转录法能高效、快速、大量合成siRNA,针对hTERT及hTR基因的siRNA可明显降低Hut78细胞株的端粒酶活性.

关键词: 端粒,末端转移酶, RNA干扰, 淋巴瘤,T细胞,皮肤

Abstract: Objectives To synthesize human telomerase reverse transcriptase (hTERT)-and human tolemerase RNA (hTR)-small interfering RNA (siRNA) and investigate their effects on telomerase activity in the cutaneous T-cell lymphoma (CTCL) cell line Hut78. Methods Two types of hTERT-and hTR-siRNAs were synthesized with T7 RNA polymerase via in vitro transcription, then either mixed with Hut78 cell lysates directly or transfected into Hut78 cells by calcium phosphate co-precipitation. Telomerase activity was tested by telomeric repeat amplification and polyacrylamide gel electrophoresis. Results With T7 RNA polymerase, hTERT-and hTR-siRNAs were synthesized efficiently with a concentration of 22.4μg siRNA per 40μL siRNA reaction mix. Telomerase activity was suppressed significantly by either of the siRNAs. The inhibition rate was 87% in the cell lysate group treated with siRNA directly, and 75% in the cell group Iransfected with siRNA. Conclusions The in vitro transcription of siRNA with T7 RNA polymerase is technically simple, costeffective, and can produce siRNA in an efficient way. hTERT-and hTR-siRNA can down-regulate telomerase activity significantly in Hut78 cells.

Key words: Telomerase, RNA interference, Lymphoma,T-cell,cutaneous