简介:AIM:ToinvestigatetheeffectofhepatitisBvirus(HBV)XgeneonapoptosisandexpressionsofapoptosisfactorsinXgene-transfectedHepG2cells.METHODS:TheHBVXgeneeukaryonexpressionvectorpcDNVA3-XwastransientlytransfectedintoHepG2cellsbylipid-mediatransfection.UntransfectedHepG2andHepG2transfectedwithpcDNA3wereusedascontrols.ExpressionofHBxinHepG2wasidentifiedbyPT-PCR.MTTandTUNELwereemployedtomeasureproliferationandapoptosisofcellsin.threegroups.Semi-quantifiedRT-PCRwasusedtoevaluatetheexpressionlevelsofFas/FasL,Bax/Bcl-xL,andc-mycineachgroup.RESULTS:HBVXgenewastransfectedintoHepG2cellssuccessfully.RT-PCRshowedthatHBxwasonlyexpressedinHepG2/pcDNA3-Xcells,butnotexpressedinHepG2andHepG2/pcDNA3cells.AnalyzedbyMTT,cellproliferationcapacitywasobviouslylowerinHepG2/pcDNA3-Xcells(0.08910±0.003164)thaninHepG2(0.14410±0.004927)andHepG2/pcDNA3cells(0.12150±0.007159)(P<0.05andP<0.01).AnalyzedbyTUNEL,cellapoptosiswasmuchmoreinHepG2/pcDNA3-Xcells(980/2000)thanHepG2(420/2000),HepG2/pcDNA3cells(520/2000)(P<0.05andP<0.01).Evaluatedbysemi-quantifiedRT-PCR,theexpressionlevelofFas/FasLwassignificantlyhigherinHepG2cellstransfectedwithHBxthaninHepG2andHepG2/pcDNA3cells(P<0.05andP<0.01).Bax/Bcl-xLexpressionlevelwasalsoelevatedinHepG2/pcDNA3-Xcells(P<0.05andP<0.01).Expressionofc-mycwasmarkedlyhigherinHepG2/pcDNA3-XcellsthaninHepG2andHepG2/pcDNA3cells(P<0.05andP<0.01).CONCLUSION:HBVXgenecanimpaircellproliferationcapacity,improvecellapoptosis,andupregulateexpressionofapoptosisfactors.TheinterventionofHBVXgeneontheexpressionofapoptosisfactorsmaybeapossiblemechanismresponsibleforthechangeincellapoptosisandproliferation.
简介:AIM:RecombinedplasmidpETNF-P16wasconstructedtoinvestigateitsexpressionpropertiesinesophagealsquamouscarcinomacelllineEC9706inducedbyX-rayirradiationandthefeasibilityofgene-radiotherapyforesophagealcarcinoma.METHODS:RecombinedplasmidpETNF-P16wasconstructedandtransfectedintoEC9706cellswithlipofectamine.ELISA,Westernblot,andimmunocytochemistrywereperformedtodeterminetheexpressionpropertiesofpETNF-P16inEC9706aftertransfectioninducedbyX-rayirradiation.RESULTS:EukaryoticexpressionvectorpETNF-P16wassuccessfullyconstructedandtransfectedintoEC9706cells.TNFαexpressionsweresignificantlyincreasedinthetransfectedcellsafterdifferentdosesofX-rayirradiationthaninthoseafter0Gyirradiation(1192.330-2026.518pg/mL,P<0.05-0.01),andtheTNFαexpressionsandP16weresignificantlyhigher6-48hafter2GyX-rayirradiation(358.963-585.571pg/mL,P<0.05-0.001).NoP16expressionwasdetectedinnormalEC9706cells.However,therewasstrongexpressioninthetransfectedandirradiationgroups.CONCLUSION:X-rayirradiationinductioncouldsignificantlyenhanceTNFαandP16expressioninEC9706cellstransfectedwithpETNF-P16plasmid.Theseresultsmayprovideimportantexperimentaldataandtherapeuticpotentialforgene-radiotherapyofesophagealcarcinoma.