小鼠IL-21 cDNA克隆及其在腫瘤基因治療中的初步研究.pdf_第1頁
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1、東南大學碩士學位論文小鼠IL21cDNA克隆及其在腫瘤基因治療中的初步研究姓名：陳國兵申請學位級別：碩士專業(yè)：免疫學指導教師：竇駿20040616——一墨莖塑望：Cloningofmurineinterleukin21cDNAandpreliminarystudyonitsapplicationintumorgenetherapyABSTRACTByChenGuobingSupervisedbyProfDollJanDepartment

2、ofMicrobiologyandImmunologyBasicMedicalSchool，SoutheastUniversityObjectiveTocloneroutineinterleukin21(rolL21)gene，constructingitseukaryocyticexpressionplasmidandapplyingitinmurineSp2／0subcutaneoustumormodelwithgenetherap

3、ytoevaluateitsanti。tumoreffectandpossiblemechanismsMethodsmlL21eDNAisamplifiedfromConAactivatedmurineTcellsbyRTPCRandthentheeDNAisclonedintoeukaryocyticexpressionplasmidpcDNA31toformrecombinantplasmidpeDNA3I，H1IL一2IThere

4、combinantplasmidistransfeetedintoSp2／0vialipofectinafteridentificationbyrestrictionenzymedigestingandDNAsequencingTheexpressionofmlL一21isdetectedbyRT_PCRandWesternBlotTheactivityoftheproteinisidentifiedaccordingtoitseffe

5、ctsoheitherthestimulatedco—proliferationofmouse丁cellsOFtheenhancedcytotoxicityofNKwhicharemeasuredbyHlTdRandMTTcolorimetryrespectivelyAfterestablishingtumoranimalmodelbyinoculatingSp2／0scintoBALB／cmice，therecombinantplas

6、midisinjecteddirectlyinloIumptissueinvivoTumorformationratetumorweighttheactivitiesofCTL，NKandLAKcells，andthelevelsofantibodyandcy￡okinesjntheserumofallmicearemeasuredforevaluatingtheanti，tumoreffectofmlL一21andilspossibl

7、emechanismsResultsThemlL一21cDNAissuccessfullyamplifiedfromtheConAactivatedmurineTcellsThoughtherearethreebasesdifierencebetweenthesequenceacquiredbyusandthesequenceofGerd3ankaccessionnumberAF254070，theaminoacidsequenceco

8、dedproteinshasnochangeoursequencehasbeenacceptedbyGenBankwiththeaccessionnumberAY428162111erecombinantplasmidpcDNA31／mlL21isconstructedcorrectlyandtheexpressionofgeReandproteinofrolL一2Iisdetectedintransformedsp2／0cellsTh

9、eexpressionproteincanco—stimulatethemurineTcellsproliferatingandenhancethecytotoxicityofNKcellsinvitroThesubcutaneoustumormodelinmouseisestablishedSuccessfullyAftertreatmentwiththerecombinantplasmid，thetumorgrowthissuppr

10、essed；theactivitiesofimmunecellsareimproveddramatically，suchasCTLandNKThelevelof1FN7ishigherthanthatofcontrolmicebutnodifierencesisfoundintheLAKactivityandantibodyleveibetweenexperimentandcontr01miceConclusionThecloningo

11、fmlL2IeDNAjssuccessfulandtherecombinantplasmidpcDNA31／mIL一21hasalsobeenconstructedSUCCESSfullywhichcanexpressmlL21proteinwithbio—activityThegenetherapyofroll。一21cansuppressthetumorgrowthinmurinetumormodelTheactivityofthe

12、cell—mediatedimmunityintumorbearingmiceisimproveddramatically，especiallyNKandCTLactivitiesTheseresultssuggestedthatpcDNA31／mlL2Iinoculateddirectlyintotumorisapotentiatapproachforgenetherapyanditsmechanismmayassociatewith

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