小分子干扰RNA真核表达载体阻断U937细胞肿瘤坏死因子α基因的表达  

Blocking effect of vector-based small interfering RNA on tumor necrosis factor-alpha gene expression in human U937 cells

作　　者：孙克宁[1] 高希武[1] 王真[1] 金群华[1] 

机构地区：[1]宁夏医科大学附属医院骨科三病区,宁夏回族自治区银川市750004

出　　处：《中国组织工程研究与临床康复》2010年第30期5635-5639,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:无菌性松动是导致人工关节置换后失败和降低人工关节使用寿命的主要原因,肿瘤坏死因子α是一个极具吸引力的无菌性松动治疗新靶点。小分子干扰RNA作为一种新型的基因阻断技术,被广泛用于新基因筛选、基因功能鉴定、信号转导研究以及基因治疗方面,显示出广阔的应用前景。目的:构建人肿瘤坏死因子α基因的小分子干扰RNA真核表达载体,观察其对U937细胞中肿瘤坏死因子α基因表达的干涉作用。方法:将合成的2对小分子干扰RNA寡核苷酸链分别退火形成双链,连接入pSilencer4.1-CMVneo真核表达载体,分别命名为pSilencer-T1和pSilencer-T2,经酶切及测序鉴定。电转染法转染重组质粒入U937细胞,G418筛选后反转录-聚合酶链反应检测其对肿瘤坏死因子α基因mRNA的干涉效果。结果与结论:经酶切及测序鉴定,成功构建了siRNA真核表达载体,经电转染法转染U937细胞后,反转录-聚合酶链反应显示所构建的干涉肿瘤坏死因子α基因的真核表达载体成功地抑制了目的基因的表达,U937细胞中肿瘤坏死因子α基因的mRNA表达水平明显降低。提示成功构建了人肿瘤坏死因子α基因的RNA干涉真核表达载体pSilencer-T1和pSilencer-T2,并在U937细胞中有效地发挥了对肿瘤坏死因子α基因表达的干涉作用。BACKGROUND：Aseptic loosening of the prosthesis has become the most common complication of total joint replacement and reduces prosthesis lifespan.Tumor necrosis factor-α（TNF-α） is a novel target for treating aseptic loosening.Small interfering RNA（siRNA） as a novel gene blocking technique has been widely used in gene screening,identification,signal transduction and gene therapy.OBJECTIVE：To explore the blocking effect of siRNA on the expression of TNF-α gene in U937 cell line using siRNA eukaryotic expression vector.METHODS：Two TNF-α siRNA cDNAs were synthesized according to the TNF-α gene sequence and cloned into the vector pSilencer4.1-CMV-neo and named pSilencer-T1 and pSilencer-T2,respectively,which were further identified by restriction endonuclease digestion analysis and DNA sequencing.U937 cells were transfected with pSilencer-T1 and pSilencer-T2 by electrotransfer.After G418 selection,the cells were selected,and the interfering effect was detected by RT-PCR.RESULTS AND CONCLUSION：Restriction endonuclease digestion analysis and DNA sequencing results showed that the target segments were cloned into pSilencer4.1-CMV neo vector respectively.The results of RT-PCR indicated that both siRNA vectors could successfully knock down TNF-α gene expression.The vector-based siRNA on TNF-α gene can effectively knock down TNF-α gene expression.Results showed that pSilencer-T1 and pSilencer-T2,human TNF-α gene siRNA eukaryotic expression vector were successfully constructed and blocked TNF-α gene expression.

关 键 词：无菌性松动 肿瘤坏死因子Α 小分子干扰RNA 真核表达载体 U937细胞 

分 类 号：R318[医药卫生—生物医学工程]