组蛋白乙酰化酶RNAi慢病毒文库构建及其对胆囊癌细胞的感染  

Construction of histone acetyltransferases RNAi lentivirus liabray and infection of gallbladder cancer cells

作　　者：杨超[1] 季福[1] 沈火剑[1] 朱宏毅[1] 李甫[1] 何敏[1] 陈涛[1] 李可为[1] 王坚[1] 施维锦[1] 

机构地区：[1]上海交通大学医学院附属仁济医院普外科,上海200127

出　　处：《肝胆胰外科杂志》2012年第3期224-227,共4页Journal of Hepatopancreatobiliary Surgery

基　　金：上海交通大学医学院胆道特色专科基金(101005144)

摘　　要：目的构建人基因组中16个组蛋白乙酰化酶的短发卡RNA(shRNA)慢病毒载体文库,为进一步研究表观遗传因子对胆囊癌的调控机制提供有利的工具。方法依据shRNA引物设计原则,分别针对每个基因设计4对shRNA引物,将引物退火形成粘性末端后连接至慢病毒载体;经菌落PCR、酶切验证正确后,进行转化及质粒抽提。结果构建了16个组蛋白乙酰化酶基因的shRNA慢病毒载体,共64个shRNA慢病毒载体克隆;并对胆囊癌细胞SGC996和GBC-SD进行了病毒感染的MOI值(multiplicity of infection)摸索,以确保可通过RNAinterference(RNAi)慢病毒干扰的方式对这两株细胞进行研究。结论成功构建了16个组蛋白乙酰化酶的shRNA慢病毒载体,从而为在SGC996和GBC-SD胆囊癌细胞中研究人组蛋白乙酰化酶奠定坚实的基础。Objective To conduct sixteen small hairpin RNA（shRNA） lentivirus vectors targetinghistone acetyltransferases and provide an advantageous tool for researching the mechanism of epigenetic factors regulating gallbladder carcinoma. Methods Following the principle of shRNA primers rules, four pairs primers for per genes were designed. After synthesized , the primers were annealed to form a cohesive ends and linked with the lentivirus empty vectors. Then identified by Colon,/PCR and digests, the positive clones were transformed and the plasmid were extracted. Results Sixteen shRNA lentivirus vectors of histone acetyltransferases were constructed, sixty-four gene clones in all. The optimal MOI values of gallbladder cancer cells SGC996 and GBC-SD had be detected by empty lentivirus, the results indicated that these two cells could be infected by lentivirus at moderate MOI and then could be studied by lentivirus RNAi. Conclusion Sixteen shRNA lentivirus vectors of histone acetyltranslerases are successfully ennducted, thus laving a solid foundation for the study functional mechnism of epigenetic factors in gallbladder carcinoma.

关 键 词：组蛋白乙酰化酶 胆囊癌 RNA干扰(RNAI) 短发卡RNA(shRNA) 慢病毒载体 

分 类 号：R735.8[医药卫生—肿瘤]