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1、人Snail基因RNAi慢病毒載體的構(gòu)建與鑒定陳麗，劉求真，邱際華，焦鋒，姚開泰(南方醫(yī)科大學(xué)腫瘤研究所，廣東廣州510515)摘要:目的構(gòu)建人Sna訂基因慢病毒干擾載體及觀察干擾前后對鼻咽癌5?8F細(xì)胞增殖和侵襲的影響。方法設(shè)計Snail基因特異性RNAi靶序列，克隆至經(jīng)雙酶切后的plVTHM線性化載體，測序鑒定正確后，用病毒上清感染鼻咽癌細(xì)胞5?8F,流式細(xì)胞儀分選獲得穩(wěn)定干擾Snail基因的細(xì)胞亞系。熒光定量PCR檢測mRNA表達(dá)情況；MTT法和體外侵襲實驗分別測定對細(xì)胞增殖和侵襲的影響。結(jié)果plVTHM-siSnail慢病
2、毒干擾載體構(gòu)建正確。熒光定量PCR結(jié)果表明分選后的細(xì)胞SnailmRNA水平表達(dá)顯著降低。干擾后的細(xì)胞增殖減慢，穿透基膜的細(xì)胞數(shù)量減少，差異具有統(tǒng)計學(xué)意義(P<0.O5)o結(jié)論plVTHM-siSnail表達(dá)質(zhì)?？娠@著下調(diào)Snail基因在5-8F中的表達(dá)，在一定程度上抑制鼻咽癌細(xì)胞的增殖和侵襲。關(guān)鍵詞:Snail;RNA干擾；鼻咽癌；增殖；侵襲中圖法分類號:[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基金項目：廣東省科技計劃項目(2007B031515006)SupportedbythePlannedScienceandTechnologyprojectofGuangdongprovince,China作者簡介：陳麗(1980-),女，湖北省隨州市人，漢族，在讀碩士研究生，主要從事腫瘤轉(zhuǎn)移機制方面的研究。電話：(020)62789443,E-mail:gmyychl@163.com通信作者：姚開泰?
6、電話：(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
