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论文摘要

转化生长因子-β1shRNA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;吴军正;郭富平;朱秀丽;温德升

Author:WANG Jing, WU Jun-zheng, GUO Fu-ping, ZHU Xiu-li, WEN De-sheng

收稿日期:2006-04-25          年卷(期)页码:2006,24(02):113-

期刊名称:华西口腔医学杂志

Journal Name:West China Journal of Stomatology

关键字:短发夹状RNA,核表达载体,化生长因子-β1,

Key words:shorthairpinRNA,ukaryoticexpressionvector,ransforminggrowthfactor-β1,

基金项目:

国家自然科学基金资助项目(30371551)

中文摘要

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。

英文摘要

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-β1 expression in salivary gland mucoepidermoid carcinoma cells.

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