中文摘要：

目的构建针对细胞凋亡抑制蛋白14-3-3ζ基因的特异性RNA干涉载体，并建立稳定转染该干涉载体的人胶质瘤细胞系。方法根据14-3-3ζ编码序列，设计并合成针对14-3-3ζ基因的特异性RNA干涉片断，并将其克隆入pSilencer3．1．H1neo干涉载体中，构建14-3-3ζ基因shRNA真核表达载体pSilencer-14-3-3ζ。重组载体pSilencer-14-3-3ζ和pSilencer3．1-Hlneo阴性对照载体pSilencer3．1-HlneoNegative经脂质体LipofectamineTM2000介导法转染胶质瘤细胞株U251；转染细胞经过G418筛选，采用Westernblot方法分别在蛋白水平检测、筛选G418抗性的克隆细胞。结果酶切鉴定和核苷酸序列分析证实，成功构建了14-3-3ζ基因shRNA真核表达载体pSilencer-14-3-3ζ，经酶切、测序鉴定证实克隆正确。获得了稳定转染pSilencer-14-3-3ζ载体的胶质瘤细胞株。结论14-3-3ζ基因特异性RNA干涉载体能够显著抑制14-3-3ζ基因在U251细胞中的表达，这为进一步研究14-3-3ζ在胶质瘤细胞系U251中的生物学功能和作用机制奠定了实验基础。

英文摘要：

Objective To construct the specific RNA interference vector targeting human 14-3-3ζ gene and to establish a human glioma cell line stably transfected with the vector. Methods According to 14-3-3ζ DNA coding sequence, the specific RNA interference （RNAi） fragments targeting 14-3-3g gene were designed and synthesized, which were cloned into pSileneer3.1-Hlneo plasmid vector, and the shRNA eukaryotic expression vector pSileneer-14-3-3ζ targeting 14-3-3ζ gene was constructed. The pSilencer-14-3-3ζ vector and negative pSilencer-14-3-3ζ vector were transfected respectively into U251 cells by LipofectamineTM 2000, and the transfected cells were selected by G418. Anti-G418 clones were isolated and Western blot methods were carried out for the identification of integration of protein levels. Results The specific shRNA eukaryotic expression vector pSilencer-14-3-3ζ targeting 14-3-3ζ gene was constructed successfully, which was identified by restriction endonuclease digestion and sequencing. Cell clones sorted out with G418 selection which had stably integrated RNAi vector targeting 14-3-3ζ gene were obtained. Conclusion Expression of 14-3-3ζ gene can be suppressed markedly by specific RNA interference vector in U251 cells which provides the experimental foundation for the further study on the biological functions and mechanisms of 14-3-3ζ in U251 cells.