细胞永生化技术及其应用研究进展

,,,..(cellim-mortalization),.,10-5~10-6,,10-12[1].,,、,,,(immortalizedcellstrains),.(telomere)(telomerase).,,.:2011-05-03;:2011-06-30:(Y2006D26):(1978-),,,,,,E-mail:[e-mail],Tel:[phone];*:(1963-),,,,,,E-mail:[e-mail].1,2,1,2,1,1*(1.,256600;2.,130062):.,.,,.、、.:;;:Q813.5:A:1007-7847(2011)04-0363-06ProgressinResearchandApplicationofCellImmortalizationTechnologyZHUANGJin-qiu1,2,MEIJian-guo1,2,WANGWen-xiu1,SHENZhi-qiang1*(1.ShandongBinzhouAnimalScience&VeterinaryMedicineAcademy,Binzhou256600,Shandong,China;2.CollegeofAnimalScienceandVeterinaryScience,JilinUniversity,Changchun130062,Jilin,China)Abstract:Thestudyoncellimmortalizationbecomesoneofthehotresearchareasofcellbiology.Manyfac-torscancausecellsimmortalization,telomeraseplaysanimportantroleintheprocessofcellsimmortaliza-tion.Thetechnologyofcellsimmortalizationwillbesuccessfullyapplicatedinmanyareasinthefuture.Itwillprovideapowerfultoolfortheresearchanddevelopmentofthebiologicalproducts,andalsogiveoutanewmethodfordiagnosisandtreatmentofdisease.Anoverviewoftheadvanceintheprinciples,methods,approachesaswellasapplicationsofthecellimmortalizationtechnologywasgiven.Keywords:immortalization;telomerase;applicationLifeScienceResearch2011154363～368··154Vol．15No．420118LifeScienceResearchAug.20112011,,.1,.,,p53、pRB,(senescence,M1),40~60,,DNA,(cell-cyclecheckpoint),DNA,,.、M1,M1.20~30(populationdoublings,PDs),(cri-sis,M2),,、,,,,,RNADNA(telom-ererestrictionfragments,TRFs),,M2,[2].,p53pRB,“”,[3].22.1,,..、,.2.1.1DNA-,(G)DNA[4].DNA,.,“”.5′-TTAGGG-3′,5~15kb.DNA、,,.,1,50~200bp[5],,,,,,.,,[6].2.1.2,RNA.1985Blackburn.,RNA,DNA3′-OHDNA,,,.“”DNAG,GG-G(Watson-CrickG-C),,DNA5′.1989Morin,:RNA(telomeraseRNA,TR)、(telomerase-associatedprotein,TEP)(telomerasereversetranscrip-tase,TERT)(telom－erasecatalyticsubunit,TCS)[7].,,[8].,[9].(hTERT),[6],hTERT.,,.pH6.5~8.5,364435~40℃,l~25mmol/L.2.22.2.12.2.1.1EBEB(epstein-barrvirus)B,.EBV100,EBVB,.,:EBNA(EBNA1、EBNA2、EBNA-LP、EBNA3)、LMP(LMP1、LMP2A、LMP2B)RNA(EBER1、EBER2).EBNA1、EBNA2;EBNA-LPpRBp53,EBNA-LPB;EBNA3A、EBNA3C、LMP-1B;EBNA3B,CD40.EBV,p53,,,EBVp53,.EBVB,B.,EBB,EB,.2.2.1.2SV40SV40,SV40T,,.Shay(1989),,SV40,1/(105~108).SV40,Aboagye(1996)SV40Tp53p53.Scrinivasan(1997)SV40T,TpRB-E2F,E2FpRB-LT,[10].SmallSV40,.,SV40.,SV40,:SV40、、.2.2.1.3(humanpapillomavirus,HPV)、、,10-5[11].HVBE6,hTERT;hTERHBVhTER,hTERT[12].Klingelhutz[13]HPV1618E6E7,M2TRFs,30~100bp,M2,TRFs.Fu-rukawa(1996)E6E7,20,.E1A[14].2.2.2,.,,Myc、c-Jun、c-Ias、vsrcMdm2,.Myc,Myc.:TERTMyc,TERT.,MycTERTmRNA,,[15].2.2.3p53pRB,p53pRB2.CDKN2A,INK4A(p16),ARF(p14,p19).p163652011,cyclinD14(cyclin-dependentkinase,CDK4)6(CDK6),pRB[16];ARFMDM2,p53[17].p53pRB.p53,p53.()p53.273p53[18].,239p53.,INK4AARF,,INK4A,ARFp53[19];INK4A[20];INK4A,,[21];INK4ARAS[22],INK4A.p53pRB[23].:、p53pRB,ARF-p53.2.2.4X、、60Co,.,.O'keilly(1997)2,2[24].2.3,、.,、,,.、、.,.、[25].31998,BodnarhTERT,,,β-().,20PDs.,,PDs2~3[26].WangJhTERT,,40PDs,β-,PAI().,IMR90hTERT,,10kb(IMR907.5kb),124PDs,57~58PDs..VazirihTERT10~15PDs(BJ),,124PDs,28PDs.,hTERT(humanchromosomeinstabili-tysyndromes)/.,CD8+T、.,、[27].,.,,[28].,40~60kb,10kb[29].,.3664125,310[30].,.,.,.4,,..4.1Southern-blot,Northern-blotWestern-blot.,.4.2,.PCR(TRAP-PCR).,,.4.3,,(、PAPL、SAB、SPA).4.4,;、.,,,.5、,,,,.,、,、,,.,,“”,Ml.,、,,,.,,,,,,.,,p53p21.,pRB(hyperphosphoryla-tion),(con-fluent)(hy-pophosphorylation),p16INK4A,[31].,TERTM1M2,,.M1,,[32].hTERT,,,.,,,.,,,、.,、、,3672011.,,,.(References):[1]KATAKURAY,ALAMS,SHIRAHATAS.Immortalizationbygenetransfection[J].MethodsinCellBiology,1998,57:69-91.[2]HABERDA.Telomeres,cancerandimmortality[J].NewEng-landJournalofMedicine,1995,332(14):955.[3]HAHNWC,WEINHERGRA.Modellingthemolecularcir-cuitryofcancer[J].NatureReviewsCancer,2002,2(5):331-341.[4]BLACKBURNEH.Structureandfunctionoftelomeres[J].Nature,1991,350(6319):569-573.[5]LANGET.Protectionofmammaliantelomeres[J].Oncogene,2002,21(4):532-540.[6]CEMIC.Telomeres,telomerase,andMyc.Anupdate[J].MutationResearch-reviewsinMutationResearch,2000,462(1):31-47.[7]GREIDERCW,BLACKBURNEH.ThetelomereterminaltransferaseofTetrahymenaisaribonucleoproteinenzymewithtwokindsofprimerspecitlcity[J].Cell,1987,51(6):887-898.[8]MASUTOMIK,YUEY,KHURTSS,etal.Telomerasemain-tainstelomerestructureinnormalhumancells[J].Cell,2003,114(2):241-253.[9]PFITZENMAIERJ,ELLISWJ,ARFMANEW,etal.Telom-eraseactivityindisseminatedprostatecancercells[J].BJUIn-ternational,2006,97(6):1309-1313.[10]SRINIVASANA,MCCLELLANAJ,VARTIKARJ,etal.Theamino-terminaltransformingregionofsimianvirus40largeTandsmalltantigensfunctionsasaJdomain[J].MethodsinCellBiology,1997,17(8):4761-4773.[11]FURUKAWAT,DUGUIDWP,ROSENBERGL,etal.Long-termcultureandimmortalizationofepithelialcellsfromnor-maladulthumanpancreaticducttransfectedbytheE6E7geneofhumanpapillomavirus16[J].AmericanJournalofPathol-ogy,1996,148(6):1763-1770.[12],,.[J].(YANGNa-na,WANGJuan,DULi-xin.Researchprogressinthecellimmortalization[J].ChinaAnimalHusbandry&Vet-erinaryMedicine),2005,32(1):37-39.[13]KLINGELHUTZAJ,BARBERSA,SMITHPP,etal.Restorationoftelomeresinhumanpapillomavirus-immortalizedhuman