治疗性克隆研究的现状与展望PPT课件.ppt

治疗性克隆研究的现状与展望,Currentstatusandprospectoftherapeuticclonestudy,Islettransplantationinsevenpatientswithtype1diabetesmellitususingaglucocorticoid-freeimmunosuppressiveregimen,A.M.JamesShapiro,etal.UniversityofAlbertaHospital,Edmonton,Canada,NewEnglJMed2000，343(4):230-238,核心内容如下：胰岛分离纯化后即刻进行移植移植足够数量的胰岛组织（两名或以上供体）抗免疫排斥反应采用非类固醇激素的药物和单克隆抗体,胰岛移植示意图,Edmonton方案的最新进展,国际多中心临床试验结果显示：一年以上不依赖胰岛素治疗者达64%三年以上不依赖胰岛素治疗者达53%血清中能检测到C肽者达72%值得一提的是，有四家开始参与该研究的中心未能获得任何一例的成功胰岛移植是治愈糖尿病的潜在希望,ADAannualMeeting,2004,Orlando,胰岛移植面临的两大挑战,胰岛组织来源：远远满足不了需要免疫排斥反应：移植失败的重要原因,DefinitionofStemCells,Self-renewalMulti-lineagedifferentiation,Cornea,Pancreas,Nerve,Liver,Skin,ClassificationofStemCells,DifferentiationcapacityTotipotentstemcellsPluripotent(multipotent)stemcellsOligopotent(unipotent)stemcellsSourceEmbryonicstem(ES)cellsSomatic(adult)stemcells,WhatisEScells?,Primitive(undifferentiated)cellsfromtheembryothathavethepotentialtobecomeawidevarietyofspecializedcelltypes.,EScells,Pluripotentstemcells,Invitrofertilization,Totipotentcells,Blastocyst,Innercellmass,(ICM),-NIHsdefinition,胚胎干细胞的研究历程,1981年，Martin从小鼠囊胚中分离出胚胎干细胞并在体外培养1995年，Thomson从恒河猴囊胚中分离并建立了第一个灵长类动物的胚胎干细胞系,MartinGR.PNAS,1981,78(12):7634-7638ThomsonJA,etal.PNAS,1995,92(17):7844-7848,1999年，杂志将干细胞研究评为世界十大科学成就之首,列在了人类基因组计划之前,里程碑,1998年，美国学者Thomson和Gearhart分别从体外受精形成的囊胚中及从5-9周龄流产胎儿的性腺脊和肠系膜中建立了人胚胎干细胞系,Thomson和Gearhart采用不同的途径成功培养干细胞的过程,ThomsonJA,etal.Science,1998,282(5391):1145-1147ShamblottMJ,etal.PNAS,1998,95(23):13726-13731GearhartJD.Science,1998,282(5391):1061-1062,Milestone,In2004,ScientistsfromKoreaandU.S.Areportedthederivationofapluripotentembryonicstemcellline(SCNT-hES-1)fromaclonedhumanblastocyst,HwangWS,etal.Science2004,303(5664):1669-1674,人胚胎干细胞的来源,Gearhart取自终止妊娠的胎儿早期性器官组织Thomson取自体外受精胚胎囊胚期的内细胞团Moon通过体细胞核转移技术获得,ThomsonJA,etal.Science,1998,282(5391):1145-1147ShamblottMJ,etal.PNAS,1998,95(23):13726-13731HwangWS,etal.Science2004,303(5664):1669-1674,Gearhart,Thomson,Moon,人胚胎干细胞(hES)的特点,形态：圆形或椭圆，体积较小，核质比较大，体外抑制分化培养时成鸟巢状，集落生长并紧密堆积，细胞界限不明显,ThomsonJA,etal.Science,1998,282(5391):1145-1147HwangWS,etal.Science2004,303(5664):1669-1674彭红梅等.北京大学学报（医学版）2004,36(6):605-608,增殖速度：1824h分裂增殖一次hES体外培养要解决的关键问题是维持细胞的分裂增殖而抑制其分化hES必须在含有白血病抑制因子（LIF）的培养基及成纤维细胞饲养层（freedercells）的条件下才能保持增殖而不分化具有分化成外、中、内三个胚层的潜能能形成嵌合体动物，从而成为联系细胞和个体之间的桥梁,ThomsonJA,etal.Science,1998,282(5391):1145-1147HwangWS,etal.Science2004,303(5664):1669-1674彭红梅等.北京大学学报（医学版）2004,36(6):605-608,人胚胎干细胞的鉴定,人胚胎干细胞具有正常稳定的二倍体核型和带型胚胎干细胞具有较高的端粒酶活性及碱性磷酸酶的表达人胚胎干细胞系端粒酶活性都很高，说明其可在体外未分化状态下进行长期培养,ThomsonJA,etal.Science,1998,282(5391):1145-1147HwangWS,etal.Science2004,303(5664):1669-1674,hES细胞有特异性表面抗原的表达,鼠和人胚胎干细胞表达的表面抗原有种属差异。Gearhart等认为SSEA-1阳性可能是源于原始生殖细胞的多能干细胞分化的标志,HwangWS,etal.Science2004,303(5664):1669-1674ThomsonJA,etal.Science,1998,282(5391):1145-1147ShamblottMJ,etal.PNAS,1998,95(23):13726-13731ThomsonJA,etal.PNAS,1995,92(17):7844-7848,具有转录因子Oct-4的表达小鼠Oct-4只限定在多潜能细胞中表达。人和小鼠的胚胎干细胞都表达Oct-4，当胚胎干细胞分化时，其表达水平大大降低Oct-4可能是哺乳动物不同发育阶段多潜能细胞所特有的少数特异性调控分子之一,ThomsonJA,etal.Science,1998,282(5391):1145-1147HwangWS,etal.Science2004,303(5664):1669-1674彭红梅等.北京大学学报（医学版）2004,36(6):605-608,人胚胎干细胞具分化的多潜能性,hES,注射,小鼠皮下,Ectoderm,Mesoderm,Endoderm,Nerve,Skin,Adrenal,Blood,Heart,Muscle22(3):265-274www.StemC,Insulinstaining,TUNEL+nuclei,DAPIstainfornuclei,Merge(A-C),Insulinstaining,FITC-Insulinuptake,InsulinstainingofEScellprogenyfrominsulinuptakeRajagopalJ,etal.Science2003,299(5605):363,Insulin/C-peptide/DNA,Insulin/Caspase-3,Insulin/DNA,TUNEL,Insulin/DNA,Insulin/FITC-Ins/DNA,ArtifactualinsulinreleasefromdifferentiatedembryonicstemcellsHanssonM,etal.Diabetes2004,53(10):2603-2609,HanssonM,etal.Diabetes2004,53(10):2603-2609,KeyMessagesfromthesetwoStudies,EScellsdifferentiatedinmediawithoutexogenousinsulindidnotstainforinsulin,anddifferentiatedEScellssubsequentlyculturedininsulin-deficientmedialostinsulinstainingAlthoughdifferentiatedcellscontainedimmunoreactiveinsulin,theydidnotcontainC-peptideVariableinsulinreleasefromthesecellsuponglucosechallengecouldbefound,butC-peptidereleasewasnotdetectedManyoftheinsulin-immunoreactivecellswereundergoingapoptosisornecrosisThesecellstookupfluoresceinisothiocyanate(FITC)-labeledinsulinfromtheculturemediumSuggestingthatcontaininginsulininthesecellsisnotasaresultofbiosynthesisbutfromtheuptakeofexogenousinsulin,RajagopalJ,etal.Science2003,299(5605):363HanssonM,etal.Diabetes2004,53(10):2603-2609,Conclusion,Insulindetectingalonecanoverestimategenuinebcelldifferentiationwhenexogenousinsulinispresent.Therefore,severalmethodsshouldbecombinedforreliableanalysisofinsulinexpressionincluding,C-peptidestainingandreleasingElectronmicroscopyNorthernblotInsituhybridizationMetaboliclabeling,Demonstrationofbi-phasicinsulinsecretionTransplantationassaysforbcellfunctionthatdemonstraterescueofthediabeticphenotypeformorethanamonth,RajagopalJ,etal.Science2003,299(5605):363HanssonM,etal.Diabetes2004,53(10):2603-2609,Whatistherapeuticcloning？,Therapeuticcloning,Patient,Cellbiopsy,Enucleatedoocyte,andfusion,Nucleustransfer,ReprogrammingDevelopmentintoblastocysts,EScellsestablished,Invitrodifferentiation,Autologoustissuegraft,Prospect,Tonormalizeallstepsa