脐血造血干/祖细胞体外扩增实验研究

EX VIVO EXPANSION OF UMBILICAL CORD BLOOD HEMATOPOIETIC STEM/PROGENI-TOR CELLS

目的:探讨人脐血造血干/祖细胞(hematopoietic stem/progenitor cells,HSC/HPC)在适合临床移植条件下体外扩增以获得理想的造血干/祖细胞数量的最佳细胞因子组合及扩增时间,为脐血经体外扩增后成为高体重受者克服脐血移植细胞数量不足及缩短植入时间提供客观指标。方法:采集脐血4份,分离单个核细胞(mononuclear,MNC),免疫磁珠分选(MiniMACS)CD+34细胞,分别在细胞因子组合为A:SCF+FL+TPO、B:SCF+FL+TPO+IL蛳3及C:SCF+FL+TPO+G蛳CSF支持的无血清、无基质的液体培养基中扩增培养14 d,收集扩增前、扩增后7 d及14 d的细胞进行细胞表型分析、半固体集落培养及端粒酶活性分析。结果:在含细胞因子IL蛳3组扩增培养7 d及14 d,均获得了总有核细胞数(total nucleated cells,TNC)及集落形成细胞(colony蛳forming cell,CFC)最大的扩增倍数。扩增7 d,IL蛳3组的CD+34细胞扩增倍数为(10.11±7.21 )倍,较其他两组稍低,但无统计学意义;三组CD+33细胞、CD+41细胞、CD+71细胞也得到了较大程度的扩增,IL蛳3组高于其他两组;3组扩增后的细胞端粒酶活性均上调,7 d时A值达最高,IL蛳3组低于G蛳CSF组(P =0.008)。随着扩增时间的延长,扩增后的细胞端粒酶活性下降;CD+34细胞占TNC的比例也明显下降,部分定向祖细胞的比例也出现先升高后?

Objectives:Toexploretheoptimalcombinationofcytokinesandtimeofexpansionum-bilicalcordblood(UCB)hematopoieticstem/progenitors(HSC/HPCs)invitrotoacquiresufficientHSC/HPCsforadulttransplantation.Methods:WehaveattemptedtoexpandselectedCD+34cellsin2蛳weekculturesinthepresencevariouscytokinecombinationsunderserum蛳freeandstromal蛳freemediumconditionsandana-lyedtotalnucleatedcells(TNC),colony蛳formingcell(CFC),telomeraseactivityandphenotypingofexpandedcellsafter7dand14d.Results:ThehighestfoldsamplificationofTNCandCFCinthepresenceofIL蛳3after7dand14dofculture.Morethan10蛳foldincreaseofCD+34cellswithIL蛳3aswellaswithoutIL蛳3after7dofculture,expansionfoldofCD+34cellsdecreaseslightlywithIL蛳3orG蛳CSFafter14dculture.Propor-tionofCD+33cells,CD+41cellsandCD+71cellsincreasedafter7dofcultureintheconditionsofthreegroupsofcytokinecombinationanddecreased14dofcultureexceptCD+41cellswithoutIL蛳3.Thetelomeraseactiv-itywasupregulatedtothehighestpointcompanyedwithstem/progenitorcellsextensivelyexpansionafter7dofcultureincludingvariouscytokinecombinations,butweredownregulatedinthelongertimeculture.TheAtitreoftelomeraseofexpandedcellswithIL蛳3waslowerthanwithG蛳CSFafter7dofculture(P =0.008).Conclusion:EnoughTNCandCD+34cellstoprovideintermediateandrapidshort蛳termengraftmentforadulttransplantationwereacquiredafter7dexpansionofafractionofUCBCD+34cellsinvitrowithcytokinessupport.TheoptimalexpansiontimewithIL蛳3is7dorso.TheabilityofIL蛳3tofacilitatedifferentiationishigherthanG蛳CSFinthepresenceofSCF,TPOandFL.