转化生长因子-β1 shRNA真核表达载体的构建  

Construction of Eukaryotic Expression Vector of Short Hairpin RNA for Transforming Growth Factor-β1

作　　者：王静[1] 吴军正[1] 郭富平[1] 朱秀丽[1] 温德升[1] 

机构地区：[1]第四军医大学口腔医学院生物教研室,陕西西安710032

出　　处：《华西口腔医学杂志》2006年第2期113-116,共4页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目(30371551)

摘　　要：目的构建转化生长因子β1(TGF-β1)的短发夹状RNA(shRNA)表达系统,为人涎腺粘液表皮样癌的治疗提供新的方法。方法根据Genebank提供的TGF-β1 mRNA序列,设计短链寡核苷酸,化学合成后经退火形成双链 DNA片段,克隆到pWH1载体中,用酶切方法对重组体进行鉴定:最后将构建的TGF-β1特异性shRNA表达载体转染涎腺粘液表皮样癌细胞,通过RT-PCR、免疫组化观察其对细胞TGF-β1 mRNA和蛋白水平表达的影响。结果经酶切、连接后构建的质粒称之为pWH1-TGF-β1。酶切证实成功构建了TGF-β1 shRNA表达载体,RT-PCR和免疫组化结果显示其能够在mRNA和蛋白水平抑制细胞内TGF-β1的表达。结论成功构建的TGF-β1特异性shRNA表达载体具有阻断TGF-β1表达的功能,可能为涎腺粘液表皮样癌治疗提供有效的方法和手段。Objective To construct the plasmid containing short hairpin RNA （shRNA） of TGF-β1 expression vector. Methods Short chain oligonucleotide was designed according to the TGF-β1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-β1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-β1 expression vector was transfected into salivary gland mucoepidermoid carcinoma （Ms） cells by Lipofectomine TM 2000, and its effect on TGF-β1 expression was observed by RT-PCR and immunohistochemistry. Results Successful construction was identified by enzyme cutting and the constructed plasmid was called pWH1- TGF-β1. The shRNA and it inhibited the TGF-β1 mRNA and protein expression effectively. Conclusion The constructed TGF-β1 shRNA expression vector can block the TGF-β expression in salivary gland mucoepidermoid carcinoma cells.

关 键 词：短发夹状RNA 真核表达载体 转化生长因子Β1 

分 类 号：R346[医药卫生—基础医学]