骨橋蛋白特異性siRNA慢病毒表達(dá)載體的構(gòu)建及其在U251膠質(zhì)瘤細(xì)胞中沉默效應(yīng)的鑒定.pdf

1、福建醫(yī)科大學(xué)碩士學(xué)位論文骨橋蛋白特異性siRNA慢病毒表達(dá)載體的構(gòu)建及其在U251膠質(zhì)瘤細(xì)胞中沉默效應(yīng)的鑒定姓名：黃智勇申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：病理學(xué)與病理生理學(xué)指導(dǎo)教師：晉雯20090501ConstructionoflentiviralvectorofsiRNAspecificforOsteopontinandcharacterizationofitsefficiencyinU251gliomacelllineAbstractobj

2、ective：ToobservetheinfluenceoflentiviralvectormediatedsmallinterferenceRNAonexpressionofhumanosteopontingeneinhumangliomacelllineU251，SOastopaveawayforOPNgenetargetedgenetherapyofgliomaEthods：Geneengineeringtechniquewasu

3、sedtoscreensmallinterferenceRNAsequencestargetingOPNgene，thesequenceswereseparatelyclonedintothepGCL—GFPvectortoconstructLV—OPNshRNA，whichwassubsequentlyconfirmedbyPCRandDNAsequencinganalysisThetiteroflentiviruswasdeterm

4、inedafter293Tcellswerecotransfected謝thLVOPNshRNA，pHelper10，pHelper20Therecombinant1entiviruseWasinjectedintoU251cellsandthemRNAandproteinexpressionwereexaminedbyRT—PCRandWesternblotting，TheproliferationofU251wasdetectedb

5、yMTTassayTheeffectsofLVOPNshRNAoninvasionofthetransfectedcellswereinvestigatedbyreconstitutedmatrigelinvasionandpolycarbonatefiltersmigrationexperimentsResult：1LentiviralsiRNAvectorsofOPNgenehavebeensuccessfullyconstruct

6、ed，2Aftertransfection、】l，ithLV—OPNshRNA，OPNexpressioninU251cellswassignificantlyinhibitedatbothmRNAandproteinlevelscompared、析ththenontransfectedandemptyvectortransfectedU251cells3AftertransfectionwitllLV—OPNshRNAMTTassay

7、showedthattheproliferationofU251cellswasSignificantlyinhibitedcomparedwitllthenontransfectedandemptyvectortransfectedU251cells4Aftertransfectionwi廿lLVOPNshRNAtheinvasionofU251cellsWassignificantlyinhibitedcomparedwiththe

8、nontransfectedandemptyvectortransfectedU251cellsConclusion：TheOPNgeneWassuccessfullysilencedinU251cellsbysiRNA，theproliferationandinvasionofU251cellswereobviouslyinhibitedAnditimpliesthatOPNgeneknockdownasanewstrategyint