稳定整合靶向性14-3-3ζ基因RNA干涉载体的胶质瘤细胞系建立  

Establishment of glioma cell line with stable RNA interference vector targeting 14-3-3ζ gene

作　　者：彭岱[1] 杨晓亮[1] 章翔[1] 曹卫东[1] 

机构地区：[1]第四军医大学西京医院神经外科,陕西西安710032

出　　处：《中华神经外科疾病研究杂志》2010年第2期132-134,共3页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(30672371);陕西省科技基金资助项目(2008K0909)

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-H1neo阴性对照载体pSilencer3.1-H1neo Negtive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Western blot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。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.

关 键 词：RNA干涉 14-3-3ζ 胶质瘤 

分 类 号：R730.54[医药卫生—肿瘤]