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

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

作　　者：徐秀莲 戚金亮[2] 陈声利 姜祎群 曾学思[1] 孙建方 

机构地区：[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

出　　处：《中华皮肤科杂志》2005年第11期685-688,共4页Chinese Journal of Dermatology

基　　金：国家教委高校博士点基金资助项日(20030023055)

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 22．4 μg／40μL siRNA合成体系。经250 ng siRNA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。30 ng siRNA转染细胞36 h后,可降低端粒酶活性20％左右,而250 ng siRNA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。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 transfected 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.

关 键 词：端粒 末端转移酶 RNA干扰 淋巴瘤 T细胞 皮肤 细胞端粒酶活性 SIRNA HTR基因 HTERT 高效合成 Hut78细胞株 聚丙烯酰胺凝胶电泳 

分 类 号：R346[医药卫生—基础医学]