PTTG基因siRNA载体的构建与鉴定  

Construction and identification of siRNA expression vectors for silencing PTTG gene

作　　者：沈云龙 崔玉婷 朱建峰 吕兰海[2] 邹俊涛[2] 郭开华[2] 武凤鸣[2] 

机构地区：[1]从化市中心医院神经外科,广东广州510940 [2]中山大学中山医学院人体解剖学教研室,广东广州510080

出　　处：《解剖学研究》2012年第4期280-283,共4页Anatomy Research

基　　金：广州市医药卫生科技项目201102A213209;广东省医学科学技术研究基金A2011529

摘　　要：目的构建针对垂体腺瘤细胞系AtT20的高效率沉默PTTG基因的shRNA表达载体。方法合成特异性干扰PTTG基因的小发卡RNA(shRNA)片段,并与pGenesil2载体连接,构建PTTG干扰载体(pGenesil2-PTTG shRNA)。利用脂质体将其转染AtT20细胞,分为正常细胞对照组、阴性对照组和3个shRNA干扰组(pGenesil2-PTTG siRNA1、2及3),应用半定量逆转录聚合酶链式反应(RT-PCR)和Western blot法分析转染后AtT20细胞中PTTG的mRNA和蛋白表达水平。结果酶切证实PTTG-shRNA表达载体构建成功;转染效率可达75%左右。转染pGenesil2-PTTG shRNA后,3个干扰组的AtT20细胞中PTTG的mRNA和蛋白表达水平均较正常对照组显著降低(P<0.01)。结论成功构建了能高效沉默PTTG的RNAi表达载体;且pGenesil2-PTTG siRNA高效地抑制了垂体腺瘤细胞AtT20细胞中PTTG綦因的表达。Objective To construct the specific high efficiency small interfering RNA （siRNA）expression vector that can silence PTrG gene. Methods Using vector based RNA interference technique, vectors were constructed to transcribe the functional short hairpinRNA （shRNA） specially targeting PTrG.The vectors were used to transfect AtT20 cells by lipofectamine2000 reagent. And the cells were divided into five groups：normal control, negative group and three siRNA interfering groups （pGenesil2-PTTG siR- NA 1, pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3）. The expression levels of mRNA and protein of PTTG were analyzed by RT-PCR and Western blotting methods. Results By restriction endonuclease and DNA sequencing analyzing, eukaryotic expression plasmid of PITG was successfully constructed, PTTG mRNA and its protein level in transfected AtT20 cells with PTrG RNAi plasmid decreased significantly compared with the normal control group （P〈0.01）, and the transfection rate reached approximately 75%. Conclusion pGenesil2-PTTG siRNA vectors were successfully constructed,and they can silence the PTrG gene in AtT20 ceils with high efficiency.

关 键 词：PTTG RNA 干扰 垂体腺瘤 

分 类 号：R736.4[医药卫生—肿瘤]