短发夹RNA对SMMC-7721肝癌细胞株ICAM-1基因表达的影响  

Suppression of CD54 expression in SMMC-7721 cell line by CD54 shRNA

作　　者：徐静[1] 陈孝平[1] 向秋[2] 廖维甲[2] 

机构地区：[1]华中科技大学同济医学院肝胆胰外科研究所,武汉430021 [2]桂林医学院,541001

出　　处：《中华实验外科杂志》2007年第1期33-34,共2页Chinese Journal of Experimental Surgery

基　　金：广西科技厅自然基金资助项目（桂科自0640121）;广西新世纪十百千人才基金项目（桂政发2003-51）;广西百名中青年学科带头人资助项目（桂教人2002-467）

摘　　要：目的探讨细胞间黏附分子-1（ICAM-1，CD54）基因的短发夹结构RNA（shRNA）表达载体pGenesil-1／CD54对SMMC-7721肝癌细胞株CD54表达的抑制作用。方法设计CD54基因shRNA片段和阴性对照shRNA片段，构建pGenesil-1／CD54和阴性对照表达载体，载体在eoli菌扩增、鉴定后，转入SMMC-7721细胞。应用免疫组织化学技术检测肝癌细胞中CD54基因的表达。结果阳性和阴性表达载体酶切，电泳有65bp和4．9kb条带，载体插入片段测序，与设计序列相同，表达载体构建正确。免疫组织化学检测表明，阴性对照表达载体转染SMMC-7721细胞，细胞膜和细胞质着黄色和棕色，pGenesil-1／CD54转染SMMC-7721细胞，不着色或着淡黄色。结论CD54的shRNA能特异性地抑制CD54在肝癌细胞SMMC-7721中的表达。Objective To study the inhibition of CD54 expression in SMMC-7721 cells by using an expression vector pGenesil-1 carrying short hairpin RNA （shRNA）. Methods shRNA DNA fragment of CD44 gene and negative contrast shRNA DNA fragment were synthesized, shRNA DNA fragments were cloned to expression vector pGenesil-1, and the negative contrast expression vector and pGenesil-1/CD54 were transfected to SMMC-7721 cells by using Lipofectamine TM2000. The protein level of CD54 in SMMC-7721 cells was detected by immunohistochemical staining. Results The negative and pGenesil-1/ CD54 expression vectors were digested by Hindlll and BamH I . There were 65bp and 4.9kb DAN bands by electromigratory analysis. The expression vectors were sequenced, and the bases and direction of the insert were correct. SMMC-7721 cells transfeeted with pGenesil-1/CD54 vector were colorless or primrose, while the cells transfected with the negative contrast expression vector were yellow or brown in cell membrane and cytoplasma. Conclusion The shRNA could induce sequence-specific CD54 gene silencing in SMMC-7721 cells.

关 键 词：短发夹RNA RNA干扰 CD54 SMMC-7721细胞 基因表达 

分 类 号：R686[医药卫生—骨科学]