摘要
Objective:To studytheeffectsof antisensehumantelomeraseRNA(ahTR)transfectionon themalignantbeha-viorsof gastriccarcinomacelllineSGC-7901anditspotentialroleingenetherapyfortumor.Methods:AnantisensehTR eukaryoticexpressionvectorcontainingthesequenceof templateregionof telomererepeatswas transfectedintogastric carcinomacelllineSGC-7901withliposomeDOTAP.Theexpressionsof hTRRNAandantisensehTRRNAwereob-servedwithRT-PCR,telomeraseactivitywithPCR-ELISA.Telomerelengthwas measuredwithSouthernblot.Cellmor-phologyandcellularproliferationcapacitywerestudiedwithMTTassay.Cellcycledistributionandapoptoticstatewere observedwithflowcytometry.Efficiencyof cloneformationin softagarandtumorigencityin nudemicewereexamined andevaluatedinahTR-transfected7901cells,andplasmidpCL-neotransfected7901cellsandparental7901cellsserved as control.Results:AnantisensehTReukaryoticexpressionvectorwastransfectedinto7901cellssuccessfully.Thetelom-eraseactivityin ahTR-transfected7901cellswas decreasedfrom100%to about25%,andtelomerelengthin thecells shortenedfrom4.08kb to3.35kb at60populationdoublings(PDs).Comparedwithparental7901andpCL-neotransfect-ed7901cells,ahTR-transfected7901cellsdisplayedsomemorphologicalchanges,includingdecreasedcellatypiaandnu-cleus/cytoplasmratiounderlightmicroscope.Furthermore,ahTR-transfected7901cellsdisplayedgrowthinhibition,de-creasedinvasivecapacityin Borden’schamberinvasivemodel,increasedG 0 /G 1 phaserateandapoptoticrate,andrestored contactinhibitionanddensityinhibition.Surprisingly,ahTR-transfected7901cellslosttheircapacityof cloneformationin softagarandcarcinogensisinnudemice.Conclusion:AntisensehTRtransfectioncaninduce7901celldifferentiationand reverseitsmalignantphenotype.Thisstudyprovidesan excitingapproachfor cancertherapythroughtheinhibitionof telomeraseactivitywithantisensegeneandothertelomeraseinhibitors.
Objective:To studytheeffectsof antisensehumantelomeraseRNA(ahTR)transfectionon themalignantbeha-viorsof gastriccarcinomacelllineSGC-7901anditspotentialroleingenetherapyfortumor.Methods:AnantisensehTR eukaryoticexpressionvectorcontainingthesequenceof templateregionof telomererepeatswas transfectedintogastric carcinomacelllineSGC-7901withliposomeDOTAP.Theexpressionsof hTRRNAandantisensehTRRNAwereob-servedwithRT-PCR,telomeraseactivitywithPCR-ELISA.Telomerelengthwas measuredwithSouthernblot.Cellmor-phologyandcellularproliferationcapacitywerestudiedwithMTTassay.Cellcycledistributionandapoptoticstatewere observedwithflowcytometry.Efficiencyof cloneformationin softagarandtumorigencityin nudemicewereexamined andevaluatedinahTR-transfected7901cells,andplasmidpCL-neotransfected7901cellsandparental7901cellsserved as control.Results:AnantisensehTReukaryoticexpressionvectorwastransfectedinto7901cellssuccessfully.Thetelom-eraseactivityin ahTR-transfected7901cellswas decreasedfrom100%to about25%,andtelomerelengthin thecells shortenedfrom4.08kb to3.35kb at60populationdoublings(PDs).Comparedwithparental7901andpCL-neotransfect-ed7901cells,ahTR-transfected7901cellsdisplayedsomemorphologicalchanges,includingdecreasedcellatypiaandnu-cleus/cytoplasmratiounderlightmicroscope.Furthermore,ahTR-transfected7901cellsdisplayedgrowthinhibition,de-creasedinvasivecapacityin Borden'schamberinvasivemodel,increasedG 0 /G 1 phaserateandapoptoticrate,andrestored contactinhibitionanddensityinhibition.Surprisingly,ahTR-transfected7901cellslosttheircapacityof cloneformationin softagarandcarcinogensisinnudemice.Conclusion:AntisensehTRtransfectioncaninduce7901celldifferentiationand reverseitsmalignantphenotype.Thisstudyprovidesan excitingapproachfor cancertherapythroughtheinhibitionof telomeraseactivitywithantisensegeneandothertelomeraseinhibitors.
基金
SupportedbyNationalNaturalScienceFundationofChina(No.39770302)
