慢病毒载体介导的人肝癌HepG2细胞BC047440基因沉默钟扬，李靖，黄小兵，郑璐，杨彤翰，赵弘智，梁平（400037重庆，第三军医大学新桥医院肝胆外科）([摘要]目的构建BC047440基因RNAi慢病毒干扰载体，并检测其对人肝癌细胞系HepG2细胞中BC047440基因的沉默效果。方法针对已经筛选确定的BC047440基因RNAi有效靶序列，合成BC047440基因特异性shRNA序列，退火形成双链DNA，与经HpaI和XhoI双酶切后的pGCSIL-GFP载体连接产生GC-shBC047440慢病毒载体，PCR筛选、鉴定阳性克隆。用脂质体转染法将GC-shBC047440、pHelper1.0载体和pHelper2.0载体共转染293T细胞,产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的表达水平测定病毒滴度，并测定其对人肝癌细胞株HepG2细胞最适感染复数（MOI）。以最适MOI感染HepG2细胞，分BC047440-shRNA组、control-shRNA组和HepG2组；RT-PCR和Westernblot检测各组细胞BC047440mRNA及蛋白的表达差异。结果经PCR鉴定，成功构建BC047440基因RNAi慢病毒载体。测定慢病毒滴度为5×108TU/ml，其在HepG2细胞中最适MOI为20；RT-PCR和Westernblot分别显示BC047440-shRNA组中BC047440mRNA及BC047440蛋白表达较control-shRNA组和HepG2组明显降低(P<?)，control-shRNA组与HepG2组间无明显差异(P>?)。结论：成功构建BC047440特异性RNAi慢病毒载体，感染人肝癌细胞系HepG2细胞后，实现了对HepG2细胞中BC047440基因的有效沉默。[关键词]：BC047440；RNA干扰；慢病毒载体；人肝癌细胞系HepG2[中图法分类号]：R735.7[文献标识志码]：ABC047440silencinginHepG2cellsmediatedbylentiviralvectorZhongYang,LIJing,HuangXiao-Bing,ZhengLu,YangTong-han,ZhaoHong-Zhi,LANGPing.(DepartmentofHepatobiliarysurgery,XinqiaoHospital,ThirdMilitaryMedicalUniversity,Chongqing400037)[Abstruact]:Objective:ToconstructalentivirusvectorforRNAinterference(RNAi)ofBC047440gene,anddetectthesilenceeffectofBC047440geneinHumanhepatocellularlivercarcinomacellline(HepG2).Methods:ThetargetingsequenceofBC047440genewhichcanbeeffectivelysilencedinRNAinferencewasconfirmedinourpreviousstudy.ThecDNAcontainingboth(基金项目：国家自然科学基金(编号：30570843)作者简介：钟扬，男，四川省泸州市人，在读硕士研究生，主要从事肝癌相关基因方面研究。E-mail:zqloove@163.com通信作者：李靖，电话：(023)-68774606，E-mail:xqyylijing@tom.comsenseandantisenseOligoDNAfragmentsofthetargetingsequencewasdesigned,synthesizedandclonedintothepGCSIL-GFPvectorwhichhavebeendoubledigestedbytheHpaIandXhoI,thenconfirmedbyPCR.293TcellswerecotransfectedwithlentiviralvectorGC-shBC047440,pHelper1.0andpHelper2.0byLipofectamine2000,producinglentiviralvectorwhichcaninfectothercelllines.ThevirustiterandthefittestMultiplicityOfInfection(MOI)inHepG2weremeasuredbytheexpressionlevelofgreenfluorescentprotein(GFP).TheinfectedHepG2wasdividedintoBC047440-shRNAgroup,control-shRNAgroupandHepG2group.ThechangesofBC047440mRNAandproteinlevelweredetectedbyRT-PCRandwesternblotrespectively.Results:ThedataofPCRdemonstratedthatthelentivirusRNAivectorofBC047440wasconstructedsuccessfully.Thetiterofviruswas5×108TU/ml,thefittestMOIinHepG2is20.BC047440andBC047440mRNAexpressionofBC047440-shRNAgroupwasobviouslowerthanthecontrol-shRNAgroupandHepG2group,buttherewasnovisibledifferentbetweenthecontrol-shRNAgroupandHepG2group.Conclusion:ThelentivirusRNAivectorofBC047440hasbeenconstructedsuccessfully.AftertheinfectionofHumanhepatocellularlivercarcinomacellline(HepG2)withthislentivirusvector,thetargetofsilencingtheexpressiononBC047440wasachieved.KeyWords:BC047440;RNAi;lentivirusvector;Humanhepatocellularlivercarcinomacellline(HepG2).Surpportedby(补充基因英文)原发性肝癌是世界上最常见的恶性肿瘤之...