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1、人Snail基因RNAi慢病毒载体的构建与鉴定陈丽，刘求真，邱际华，焦锋，姚开泰(南方医科大学肿瘤研究所，广东广州510515)摘要:目的构建人Sna订基因慢病毒干扰载体及观察干扰前后对鼻咽癌5・8F细胞增殖和侵袭的影响。方法设计Snail基因特异性RNAi靶序列，克隆至经双酶切后的plVTHM线性化载体，测序鉴定正确后，用病毒上清感染鼻咽癌细胞5・8F,流式细胞仪分选获得稳定干扰Snail基因的细胞亚系。荧光定量PCR检测mRNA表达情况；MTT法和体外侵袭实验分别测定对细胞增殖和侵袭的影响。结果plVTHM-siSnail慢病
2、毒干扰载体构建正确。荧光定量PCR结果表明分选后的细胞SnailmRNA水平表达显著降低。干扰后的细胞增殖减慢，穿透基膜的细胞数量减少，差异具有统计学意义(P<0.O5)o结论plVTHM-siSnail表达质粒可显著下调Snail基因在5-8F中的表达，在一定程度上抑制鼻咽癌细胞的增殖和侵袭。关键词:Snail;RNA干扰；鼻咽癌；增殖；侵袭中图法分类号:[R34]ConstructionandidentificationofthelentiviralRNAinterferencevectorofhumanSnailgeneCH
3、ENLi,LIUQiu-zhen,QIUJi-hua,JIAOFeng,YAOKai-tai(InstiluteofCancerResearch,SouthernMedicalUniversity,Guangzhou510515,China)Abstract:ObjectiveToconstructarecombinantlentiviralexpressionvectorforRNAinterference(RNAi)ofhumanSnailgene,andtostudyitseffectsonproliferationandi
4、nvasionofnasopharyngealcarcinomacellline5・8F.MethodsTheeffectivesequenceofshorthairpinRNAs(shRNA)targetingSnailgenewasdesigned,andclonedintothelinearpLVTHMvector,itwasconfirmedbyDNAsequencing.5・8FcellswereinfectedwiththeviralsupernatantsandthecellswithstableSnailgenek
5、nock-downwereseparatedbyFluorescenceActivatedCellSorter(FASC).The基金项目：广东省科技计划项目(2007B031515006)SupportedbythePlannedScienceandTechnologyprojectofGuangdongprovince,China作者简介：陈丽(1980-),女，湖北省随州市人，汉族，在读硕士研究生，主要从事肿瘤转移机制方面的研究。电话：(020)62789443,E-mail:gmyychl@163.com通信作者：姚开泰•
6、电话：(020)61648225E-mail:ktyao@fimmu.comexpressionofSnailmRNAwasdetectedbyRealtimeRT-PCR.MTTandcellinvasionassaywereusedtodetecttheproliferationandinvasionof5-8FcellsafterplVTHM-siSnailtransfection.ResultsThelentivirusvectorplVTHM-siSnailwasconstructedsuccessfully.These
7、parated5-8F-plVTHM-siSnailexhibitedsignificantknock-downofSnailmRNAexpression.TheproliferationweremoreslowlyandthecellpopulationthatcutthroughMatrigelwerelessafterplVTHM-siSnailtransfection(P<0.05).ConclusionplVTHM-siSnailvectorcoulddown-regulateSnailgeneexpressioninh
8、umannasopharyngealcarcinoma5-8Fcellsandsuppressitsinvasionandproliferationtosomeextent.Keywords:Snail;RNAinterference;nasoph
