慢病毒shRNA靶向干扰RegⅣ基因胰腺癌细胞株的构建  

Establishment of pancreatic cancer cells with stable interference by lentivirus shRNA targeting RegⅣ gene

作　　者：李健[1] 胡启立[1] 徐凌[2,3] 卫巍[4] 赵严[2] 刘华[2] 汤茂春[2] 宋振顺[1] 夏小俊[2] 王兴鹏[2,5] 王锋[2] 

机构地区：[1]同济大学附属上海市第十人民医院普外科,上海200072 [2]同济大学附属上海市第十人民医院消化内科,上海200072 [3]上海交通大学医学院附属同仁医院消化内科,上海200336 [4]同济大学附属上海市第十人民医院急诊科,上海200072 [5]上海交通大学附属上海市第一人民医院消化内科,上海200080

出　　处：《复旦学报（医学版）》2016年第2期147-153,共7页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81472578);上海市科学技术委员会基金(11JC1410000)~~

摘　　要：目的构建并鉴定再生基因4(regenerating islet-derived family member 4,RegⅣ)短发夹干扰RNA(short hairpin RNA,shRNA)慢病毒载体,继而建立稳定干扰RegⅣ基因表达的胰腺癌细胞株PANC-1。方法使用聚合酶链式反应(polymerase chain reaction,PCR)检测RegⅣ基因在胰腺癌细胞株中的表达情况。构建重组靶向干扰RegⅣ基因的shRNA慢病毒表达质粒pGC-shRNA-RegⅣ,用脂质体转染的方法将载体导入胰腺癌细胞,建立稳定表达shRNA的细胞株。使用荧光定量PCR检测干扰效率。RegⅣ基因过表达载体的构建方法为:将获取的目的基因与pEGFP-RegⅣ-3FLAG过表达质粒载体分别进行双酶切,利用电泳回收双酶切产物,转化感受态细胞并进行测序验证,验证成功后,使用Western blot检测其在293T细胞中的表达强度。结果 PCR结果显示RegⅣ在PANC-1中表达最高,在BxPC-3中表达最低。测序验证pGC-shRNA-RegⅣ重组质粒构建成功。将重组质粒稳定转染入胰腺癌细胞株PANC-1后能明显抑制RegⅣmRNA表达水平。过表达载体pEGFP-N1-3FLAG-RegⅣ合成成功后进行Western blot鉴定,验证pEGFP-N1-3FLAG-RegⅣ过表达载体构建成功。过表达RegⅣ基因转染BxPC-3后,与空质粒组相比,RegⅣ表达量明显提高。结论成功建立了靶向稳定干扰RegⅣ基因表达的shRNA胰腺癌细胞株PANC-1。Objective To construct siRNA expression and overexpression vector targeting RegⅣgene and establish the pancreatic cancer cells PANC-1 with stable inhibition or stable overexpression of RegⅣ gene. Methods RegⅣ gene expression were examined in several pancreatic cancer cell lines by real-time PCR.The recombinant lentivirus pGC-shRNA-RegⅣ vector and pEGFPN1-3FLAG-Reg Ⅳ vector were constructed,and then transfected into pancreatic cancer cells by Lipofectamine2000.After pancreatic cancer cells with constant expression of the pGC-shRNA-RegⅣwere established,the interference efficiency of RegⅣ mRNA expression was detected by real-time PCR. Results There was highest expression of RegⅣ gene in the pancreatic cancer cell line PANC-1and lowest expression in BxPC-3.Recombinant lentivirus pGC-shRNA-RegⅣ vector was successfully constructed by the identification of sequencing.The recombinant vector markedly inhibited RegⅣ gene expression in pancreatic cancer cell line PANC-1.Recombinant lentivirus pEGFP-N1-3FLAG-RegⅣvector was successfully constructed by sequencing and Western blot,which markedly increased RegⅣgene expression in pancreatic cancer cell line BxPC-3. Conclusions Recombinant lentivirus vector pGC-shRNA-RegⅣ and overexpression vector pEGFP-N1-3FLAG-RegⅣ targeting RegⅣ gene was successfully constructed and pancreatic cancer cell line PANC-1 with stable siRNA expression targeting RegⅣ gene and BxPC-3 with stable over-expression were established.

关 键 词：胰腺癌 再生基因4 慢病毒 短发夹干扰RNA 

分 类 号：R576[医药卫生—消化系统]