攜帶超抗原SEA基因的選擇性增殖腺病毒治療膀胱腫瘤的體外實驗研究.pdf

1、東南大學(xué)碩士學(xué)位論文攜帶超抗原SEA基因的選擇性增殖腺病毒治療膀胱腫瘤的體外實驗研究姓名：胡建鵬申請學(xué)位級別：碩士專業(yè)：臨床醫(yī)學(xué)；外科學(xué)指導(dǎo)教師：韓從輝20110531英文摘要ObjectiveAbstractConstructionofthehTERTandregulationofHIFbidirectionalpromotergenecarryingtheSEAselectiveadenovirus，invitroobservati

2、onofSEAgeneexpressioninbladdercancercellsandstimulatelympHocytefunctionassociatedcytokinesandtheircombinationofantitumorcellsMethodsThetelomerasepromoter(hTERT)andhypoxiainduciblefactorpromoterfHIF)aredigestedtheconnecti

3、oninthedeficientadenovirusElAandE1BshuttleplasmidscquencespriortocontrolElAandElBexpressionCMVpromoterandSEASV40geneexpressionandregulationoftermination，andtheshuttlevectorbackboneplasmidliposomecotransfectedhumanembryon

4、ickidney293cellsforhomologousrecombinationtoobtaindualregulationofselectivereplicationadenovimSH2SEA一Ad，togetpositiveplaque，extracted百andviralDNA，thePCRAmplificationofthevirus，cesiumchloridegradientpurificationofadenovir

5、us，virustitcrTheviruswillgetbladderCanCercellstransfectedEJ，RTPCRdetectionofSEAmRNAexpressi—onintumorcells，immunofluorescencelocalizationSEAexpressedintumorcellsWestern—blotdeterminationofproteinexpression，dynamicobserva

6、tionunderthemicroscopewereculturedlympHocytesandtumorcells，thegrowthoflivingcells，M1vrtest，ELISAdetectionofIL2，IL4，TNFsecretionResultsApplicationofPCR，restrictioncrlzymcanalysisandDNAsequencingshowedmattheSuccessfulconst

7、ructionofshuttleplasmidp3l5CSSSEA，byhomologousrecombinationsuccessfullycarrysuperantigenSEAgenefragmentsH2SEAAdadenovirusSEART—PCRdetectionofmRNAexpressionintumorcellsagarosegelelectrophoresisshowsclearbandsat252bp；seenb

8、yimmunofluorescenceandWestern—。blotproteinmadeclear；lympHocytesandtumorcellswereculturedtumorcellsl2，24，48hoursoftheexperimentalgroupWassignificantlythanthecontrolgroup；MTTtest12，24，48hoursintheexperimentalgroupthancontr

9、olgroupthenumberoflivingcells；ELISAdetectionofIL2，IL一4，TNFsecretionwerehighel“thantheexperimentalgroupConclusion1CarrythechoiceofSEAgeneadcnovirusH2SEAAdgenewassuccessfullyconstructedandexpressed2Preliminaryvalidationoft