西安交通大学口腔医院(西安710004)

hBMP27(710004)3:27(hBMP27),,:hBMP27cDNAPTcDNA2hBMP27PCR,pAAVIRES2hrGFPDH5Α,pAAVIRES2hrGFP2hBMP27293,PEG
NaCl:,,:,hBMP27pAAVIRES2hrGFP293rAAVIRES2hBMP27,3.01011v.g
mlrAAVGFP,4:rAAVIRES2hBMP27,Constructionoftherecombinedadeno-associatedviralvectorofhBMP-7andtransfectionofdentalpulpcellsHospitalofStomatology,XianJiaotongUniversity(Xian710004)ShiJianfengLiAngRaoGuozhouetalABSTRACTObjectives:Toconstructtherecombinedadeno2associatedviralvectorofhumanbonemorphogeneticprotein27(hBMP27)andtransfectdentalpulpcells;Toexplorethenewgenetherapyforrepairingthetoothtissuedamage.Methods:ThemouldswhichcontainedhBMP27vectorwereamplifiedbyPCR.ThereclaimedproductionsandpAAVIRES2hrBMPweredigestedandintegrated,bacilluscoliDH5Αcompetentcellsweretransformed,andrecombinantplasmidpAAVIRES2hrGFP2hBMP27wasobtained.293cellswerevirus2enclodedbythecalciumphosphateprecipitationmethodandthevirusfluidwasobtainedandprocessedbychloroformprecipitatetreatment,PEG8000
NaClprecipitationandchloroformextractionforpurification.Virustiterandtransfectionofdentalpulpcellsculturedinvitrowereidentifiedbydotblothybridizationmethod.Results:Calciumphosphatecoprecipitationmethodswereusedin293cellsandadeno2associatedviralvectorrAAVIRES2hBMP27wasrecombinated.Greenfluorescentproteinexpressingreachedpeakonday6indentalpulpcellsaftertransfectionbyrAAVIRES2hBMP27andtheexpressionlastedfor4weeks.Conclusion:Therecombinedadeno2associatedviralvectorofhumanbonemorphogeneticprotein27(hBMP27)wasconstructedsuccessfully,whichcantransfectdentalpulpcellseffectively.ThehBMP27anddentalpulpcellscanbeappliedtogenetherapyresearch.KEYWORDSTransfectionCellsDentalpulpGenerearrangementBonemorphogeneticproteins(Bonemorphogeneticproteins,BMPs),2Β,,27(hBMP27)3,[1]hBMP27pAAV2IRES2hrGFP2hBMP27,,1hBMP27cDNA3720081371©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.Α;(Beyotime);Fermentas()DMEMGibco;BamHISalIT4TrizolReagentkitPCRTakara;()2hBMP27GenbankhBMP27(NM2001719),CDS,hBMP27cDNAPTcDNA2hBMP27PCRP152CGGGATCCATGCACGTGCGCTCACTGCG23BamHI,P252GCGTCGACCTAGTGGCAGCCACAGGCCCGGACC23SalIPCR:942min,9430s,5330s,7230s,35,3pAAVIRES2hrGFP2hBMP27pAAVIRES2hrGFPPCR,BamHI+SalI,16T4DH5Α,(+),3718h,LB,,pAAVIRES2hrGFP2hBMP274rAAV293,3060min,,DMEM6ml1.80(A260
280)pAAVIRES2hrGFP2hBMP27pAAV2HelperpAAV2RC10Λl(1Λg
Λl),200Λl,293375%,16h,6ml10DMEM,72h,-20,37,,,1mol
LNaCl10PEG80001[2],rAAVrAAVIRES2hBMP275rAAV,4,1105
24,375%CO280%,,(100U
ml)(100Λg
ml)DMEM3200ΛlrAAVIRES2hBMP27(MOI=1105)DMEM,2h,,10%DMEM,12hGFP1hBMP27PCR,1312bpDNA,hBMP27(1)2pAAVIRES2hrGFP2hBMP27pAAVIRES2hrGFP2hBMP27BamHI+SalI,6100bp1312bpDNA(2),hBMP27()hBMP27pAAVIRES2hrGFP,Genebank3rAAV,293,,,293,GFP,,rAAVIRES2hBMP27DNA,3.01011v.g
ml,1hBMP27PCRM:DL2000;123hBMP274720081371©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.:DNAMarker,12pAAVIRES2hrGFP2hBMP274rAAV,7d,14d70%,,rAAVIRES2hBMP274,24h,6,6rAAV84.6%,4,,,(rAAV),,4681bpDNA,repcap[3],,AAV,,rAAVAAV22,repcap,ITR(),ITR[4]AAVHelper2FreeSystem,pAAVIRES2hrGFP,hrGFP,GFP,,(Internalribosomeentrysite,IRES),CMV,AAVrepcappAAV2RC,pHelperE2AE4VARNA2933.01011v.g
mlrAAV,AAV,rAAV84.6%BMPs,[5]BMP27[6]RutherfordBMPs,,Ripamondli27I,,BMP27,,,BMP27rAAV,,,,,[7]BMPs,BMP7,,[8]hBMP27pAAV2IRES2hrGFP2hBMP27,293rAAV,GFP,rAAVBMP7,BMP7[1]HidakaC,GoodrichLR,ChenCT,etal.Accelerationofcartilagerepairbygeneticallymodifiedchondrocytesoverexpressingbonemorphogeneticprotein27[J].JOrthopRes,2003;21(4):5732583.[2],,,..,2000,45(3):207122075.[3]Chiriva2InternatiM,LiuY,SalatiE,etal.EfficientgenerationocytotoxicTlymphocytesagainstcervicalcancercellsbyadeno2asso2ciatedvirus
humanpapillomavirustype16E7antigengenetransductionintodendriticcells[J].EurJImmunol,2002,32(1):30238.[4]JosephE,Rabinowitz,SamulskiRJ.Buildingabettervector:thema2nipulationofAAVvirions[J].Virology,(79)5720081371©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[1],..:,1995:122248.[2],,,..,2003,25(3):18220.[3]LudwigCU,HartmannD,LandmannR,etal.Unalteredimmunocompetenceinpatientswithnon2disseminatedbreastcanceratthetimeofdiagnosis[J].Cancer,1985,55(8):167321678.[4]Mc2CluskeyDR,RoyAD,AbramWP,etal.Tlymphocytesubsetsintheperipheralbloodofpatientswithbenignandmalignantbreastdisease[J].BrJCancer,1983,47(2):3072309.[5]SchroderW,VeringA,StegmullerM,etal.Lymphocytesubsetsinpatientswithovarianandbreastcancer[J].EurJGynaecolOncol,1997,18(6):4742479.[6],,,...2000,34(1):18220.[7],,,..,2006,26(3):2792281.[8],,.TNK.,2006,14(9):106921071.[9],,..,1999,14(1):35237.[10]BerkelH,BirdsellDC,JenkinsH.Breastaugmentation:Ariskfactorforbreastcancer?NEnglJMed,1992,326(25):164921653.[11]BirdsellDC,JenkinsH,BerkelH,etal.Breastcancerdiagnosisandsurvivalinwomenwithorwithoutbreastimplants.PlastReconstrSurg,1993,92(5):7952800.[12]PetitJY,LeMG,MouriesseH,etal.Canbreastreconstructionwithgel2filledsiliconeimplantsincreasetheriskofdeathandsecondprimarycancerinpatientstreat