干细胞研究及其应用

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1、干细胞研究及其应用干细胞研究及其应用第1页/共81页2 In early October,a Colorado couple has a baby named Adam,whose umbilical cord stem cells could cure his six-year-oldsister Mollys Fanconi anemia.今年十月发生在美国Colorado的一个故事第1页/共81页第2页/共81页3One cell is removed from an eight-cell embryo in preparation for preimplantation genetic

2、 diagnosis.第2页/共81页第3页/共81页4Two separated blastomeres subjected to FISH analysis to check the chromosomes第3页/共81页第4页/共81页5Genetic abnormalities detected with Vysis Inc.s MultiVysion PGT technology.第4页/共81页第5页/共81页6干细胞具有自我更新能力和高度增殖以及多向分化的潜能，各种功能细胞的生成及其调控依赖于各种微环境、细胞生长因子、基质细胞、细胞外基质等多种因素的相互作用与平衡，共涉及细胞的增

3、殖、分化、发育、成熟、迁移、定居、衰老、凋亡、癌变等生命科学中的许多基本问题和研究热点第5页/共81页第6页/共81页7 胚胎干细胞（embryonic stem cells，ES细胞）是由早期胚胎内细胞团或原始生殖细胞经体外分化抑制培养，在传代过程中筛选出的具有多能性的细胞。第6页/共81页第7页/共81页8干细胞研究的历史干细胞的类型 来源于胚胎-胚胎干细胞 ESC （Embryonic Stem Cell）胚胎性生殖细胞 EGC （Embryonic Germ Cell）来源于成体-成体组织来源的干细胞 ASC （Adult-derived Stem Cell）ESC/ASC的比较干细胞

4、的特点干细胞的鉴别方法干细胞的研究热点干细胞工程-干细胞的应用干细胞应用的法律、伦理道德问题第7页/共81页第8页/共81页9 1981年Evans和Kaufrnan及Martin首次由小鼠中分离得到鼠的胚胎干细胞。至今已分离得到的胚胎干细胞物种有：金黄地鼠（1988）、貂（1993）、猪（1994，1997）、鸡（1996）、恒河猴（1995）、绒猴（1996）。分离得到人的胚胎干细胞（1998），分离得到人的胚胎生殖细胞（1998），目前不断报道成年组织来源的的干细胞。干细胞研究的历史第8页/共81页第9页/共81页10干细胞的类型来源于胚胎-胚胎干细胞 ESC （Embryonic St

5、em Cell）胚胎性生殖细胞 EGC （Embryonic Germ Cell）来源于成体-成体组织来源的干细胞 ASC （Adult-derived Stem Cell）第9页/共81页第10页/共81页11第10页/共81页第11页/共81页12 Induced differentiation of human ES-derived cells in culture.第11页/共81页第12页/共81页13ES细胞的体外培养（取自干细胞和发育生物学，叶鑫生等编，2000.4）第12页/共81页第13页/共81页14Pluripotent murine embryonic germ cel

6、ls identified by red staining第13页/共81页第14页/共81页15A phase contrast image of the pluripotent murine embryonic germ cell第14页/共81页第15页/共81页16A culture dish containing hundreds of colonies of murine embryonic germ cells.Each red-stained spot represents an individual colony comprised of hundreds or thousa

7、nds of stem cells.第15页/共81页第16页/共81页17ES细胞生物学特性1细胞形态结构及核型2 细胞的高度分化潜能3 碱性磷酸酶的表达4 胚胎阶段特异性细胞表 面抗原的表达第16页/共81页第17页/共81页18 ES细胞形态结构及核型 1。各种动物的ES细胞具有与早期胚胎细胞相似的形态结构，胞体体积小，核大，有一个或几个核仁。2。ES细胞与卵圆柱期（egg cylinder stage）胚胎外胚层和胎儿生殖峙的原始生殖细胞类似，而与 ICM细胞有差异。3。细胞中多为常染色质，胞质结构简单，散布着大量核糖体和线粒体，核型正常，保留了整倍体性质。正常ES细胞染色体正常，如发

8、生异常则其很难发育分化形成动物个体。4。ES细胞在体外分化抑制培养中，呈克隆状生长，细胞紧密地聚集在一起，形似鸟巢，细胞界限不清，克隆周围有时可见单个ES细胞和分化的扁平状上皮细胞。5。ES细胞增殖迅速，每1824 h分裂增殖 1次。6。此外，其还可以在体外进行选择、操作、冻存。冻存的细胞可在需要时随时解冻，继续培养不失其原有特性，并且来自一个克隆的细胞具有同样的特征。第17页/共81页第18页/共81页19 ES细胞的高度分化潜能 ES细胞的全能性是其区别于成 纤维细胞等体细胞的显著特点1.ES细胞在体外需在饲养层细胞上培养才能维持其未分 化状态，一旦脱离饲养层就自发地进行分化。2.在单层培

9、养时细胞自发分化成多种细胞，悬浮培养中：“简单类胚体”“囊状胚体”细胞分化物。3.ES细胞可进行诱导分化 *用RA（维生素A酸或视黄酸）作诱导90以上的ES细胞 分化为神经胶质细胞，*聚集培养的ES细胞诱导分化则可分化为有节律性收缩 的心肌细胞。*将ES细胞注射到同源动物皮下，可形成组织瘤，其细胞 组成可代表3个胚层细胞。*用ES细胞作核供体进行细胞核移植，可以得到可发育重 构胚和动物个体。第18页/共81页第19页/共81页20 碱性磷酸酶的表达 许多资料表明，小鼠、大鼠的桑格胚细胞和囊胚细胞均有碱性磷酸酶（AKP）表达，小鼠的EC细胞和ES细胞中均含有丰富的AKP。而在已分化的EC细胞和E

10、S细胞中AKP呈弱阳性或阴性。猪、兔的桑格胚和早期囊胚AKP呈阳性。因此，AKP常用来作为鉴定EC细胞或ES细胞分化与否的标志之一。第19页/共81页第20页/共81页21胚胎阶段特异性细胞表面抗原的表达早期胚胎细胞表面均表达胚胎阶段特异性表面抗原（SSEA1）。在胚胎的原始外胚层细胞、ES细胞、EC细胞和原始生殖细胞的表面均可检测到SSEA1的表达。小鼠SSEA1的表达自8一细胞期开始，直到原始外胚层形成期。早期囊胚ICM细胞全部呈强阳性，晚期囊胚中部分ICM呈强阳性，部分呈弱阳性，少部分为阴性。因此，SSEA也常作为ES细胞鉴定的一个标志。第20页/共81页第21页/共81页22ES细胞的

11、鉴定-细胞的形态结构-核型分析-碱性磷酸酶（AKP）染色-SSEA-I 免疫荧光标记-分化能力检测 体外分化试验 体内分化试验 嵌合体形成试验 核移植试验第21页/共81页第22页/共81页23 ES细胞形态学鉴定 依ES细胞的形态结构（胞体体积小，核大、有一个或几个核仁）和生长特性（呈克隆状生长，细胞紧密聚集，形似鸟巢，界限不清等）对ES细胞进行初步鉴定。第22页/共81页第23页/共81页24核型分析 ES细胞具有正常二倍体核型，可用核型分析进行检验第23页/共81页第24页/共81页25 碱性磷酸酶（AKP）染色 *常用快绿（Fast Green Staining)染色法，*对照以已建系

12、ES细胞（或小鼠的脱带桑棋胚 或囊胚）作为阳性对照，*作用液中不含察酚为阴性对照。*结果判定阳性对照ES细胞染成棕色，阴性对 照无色。小鼠35日龄胚胎ICM为强阳性。第24页/共81页第25页/共81页26 SSEA-I 免疫荧光标记 第1抗体，SSEA1（鼠抗ES细胞单克隆抗体），1:100稀释；第2抗体，羊抗鼠 1：10稀释;封闭液，羊抗兔lgG。步骤杜氏PBS洗3次,2 Triton作用20 min；再以杜氏PBS洗3次,封闭液中置20min；以杜氏PBS洗3次，加第1抗体于4过液；以杜氏PBS洗3次，加第2抗体于30温箱置 30min；再以杜氏PBS洗3次后用甘油PBS封片。样本在激光

13、共聚焦图像系统下观察,结果依细胞 是否出现荧光作出判断。第25页/共81页第26页/共81页27 分化能力检测体外分化试验体内分化试验嵌合体形成核移植第26页/共81页第27页/共81页28胚胎干细胞的制备1.直接由动物体内获取囊胚再分离ICM的方法，这样得到的囊胚数量少，质量也可能不高，由此会影响分离ES细胞的效率。2.卵细胞去核培养、转入体细胞核，体外培养，获得高质量的去透明带囊胚，由此囊胚进一 步分离得到胚胎干细胞。第27页/共81页第28页/共81页29第28页/共81页第29页/共81页30第29页/共81页第30页/共81页31目前已证明，小鼠、兔、羊。猪、牛等多种动物的桑塔胚以前

14、以及囊胚的ICM细胞具有发育的全能性，因此桑植胚或囊胚成为分离ES细胞的常用材料。ES细胞的分离早期胚胎的获取及处理方法原始生殖细胞的获取分化抑制物的选择,包括：饲养层（feeder lopr），条件培养基（conditioned medium，CM）和分化抑制因子（differentiation inhibitory activity，DIA）基础培养基的选择添加物的选用第30页/共81页第31页/共81页32干细胞的研究热点人体干细胞的建株；干细胞保持不分化的培养；干细胞的定向诱导分化；干细胞应用的法律、伦理道德问题；干细胞、组织工程、器官工程；胚胎组织的种植及定向诱导分化。第31页/共8

15、1页第32页/共81页33胚胎干细胞（embryonic stem cell，ES细胞）也可被称为多能干细胞（pluripotent stem cell，PSC），是胚胎或原始生殖细胞经体外抑制分化培养后筛选出的具有发育全能性的细胞。其可以定向诱导分化为几乎所有种类的细胞，甚至形成复杂的组织和器官，其建系、体外扩增、走向诱导分化、调控机制、细胞性能、组织重构等方面的研究将在未来的人体发育、基因功能、药物开发、细胞治疗和组织器官替代治疗中发挥重要作用，并成为组织器官移植的新资源目前胚胎干细胞的研究第32页/共81页第33页/共81页34第33页/共81页第34页/共81页35Alternativ

16、e models for stem cell deployment.(A)Invariant asym-metry.A stem cell(S)gives rise by asymmetric division to a prog-enitor(P)with a more restricted proliferation potential,which differentiates in response to extri-nsic cues.The stem cell phenotype is regulated by reciprocal short-and/or long-range s

17、ignaling(thick colored arrows).(B)Populational asymmetry.Stem cells give rise to daughter cells that can be either stem cells or else progenitors that differentiate along different pathways(1,2,and 3)depending on the combination of extrinsic factors to which they are exposed.ECM,extracellular matrix

18、.第34页/共81页第35页/共81页36第35页/共81页第36页/共81页37第36页/共81页第37页/共81页38Effects of cytokines on ES cells第37页/共81页第38页/共81页39成体干细胞（Adult Stem Cells）第38页/共81页第39页/共81页40The Stem Cell Cycle第39页/共81页第40页/共81页41 目前研究的成体干细胞 （Adult Stem Cells）Bone Marrow Heamatopoietic Stem Cells(HSC)Peripheral Blood Stem Cells(PBSC)

19、Fetal Liver Stem CellsUmbilical Cord Blood Stem CellsNeural Stem Cells “Studies suggest that stem cells in different adulttissues may be more similar than previouslythought and perhaps insome cases have adevelopmental repertoire close to that of ES cells.”Science,Volume 288,Number 5471,Issue of 2 Ju

20、n 2000,pp.1660-1663第40页/共81页第41页/共81页42 骨髓干细胞Bone Marrow Stem Cells，HSC，第41页/共81页第42页/共81页43造血干细胞作为干细胞研究与应用的突破口1.造血细胞是细胞增殖分化的最佳模型。2.造血干祖细胞及各系血细胞的表面标志较为清 楚，细胞表型特征可进行定量分析，且可分选。3.造血干一祖细胞增殖及向各系分化的重要诱导因 子、受体、信号转导、微环境等因素较为清楚。4.造血细胞发挥功能可相对游离，不需“生物支架”、神经、血管、外科移植等复杂的“下游”工艺，因 此便于直接应用于临床。第42页/共81页第43页/共81页44 S

21、chematic view of adult haematopoiesis.HSC,haematopoietic stem cell;CLP,common lymphoid progenitor;CMP,common myeloid progenitor.The myeloid compartmentincludes the cell types indicated;however,the termmyeloid cells in common usage usually refers to monocytes and neutrophils.第43页/共81页第44页/共81页45Stem

22、cells from bone marrow could be used to repair a variety of tissues.第44页/共81页第45页/共81页46Isolation of Primary and Immortalized CD34 Hematopoietic and Mesenchymal Stem Cells from Various Sources Stem Cells,Vol.18,No.1,1-9,January 2000第45页/共81页第46页/共81页47Previous work with mice and human had shown that

23、 adult bone marrow stem cells could form liver,as well as bone,cartilage,tendon,nerve and other tissues.1,Neil Theise et al.;Liver from Bone Marrow in Humans;Hepatology 32,11-16,July,2000.2,Malcolm Alison et al.;Cell Differentiation:Hepatocytes from Non-Hepatic Adult Stem Cells;Nature 406,257,July 2

24、0,2000.3,Susan Okie,Bone Marrow Stem Cells Offer Hope for Liver Therapies;Study of Transplants Shows a Transformation;Washington Post,June 27,2000.4,Dr.David Whitehouse,Stem cells promise liver repair;BBC,July 19,2000.5,Neil Theise et al.;Derivation of hepatocytes from bone marrow cells in mice afte

25、r radiation-induced myeloablation;Hepatology 31,235-240,January,2000.6,Bryon Petersen et al.;Bone marrow as a potential source of hepatic oval cells;Science 284,1168-1170;May 14,1999.第46页/共81页第47页/共81页48Osteoblast-Osteoclast Interactions as a Paradigm for Osteoblast-Hematopoietic Cell 第47页/共81页第48页/

26、共81页49肝脏干细胞 Fetal Liver Stem Cells第48页/共81页第49页/共81页50The oval cells response in many mammals,including humans.(Oval cells are the progeny of potentially pluripotential stem cells in liver.)第49页/共81页第50页/共81页51神经干细胞 Neural Stem CellsAn adult neural stem cell has a very broad developmental capacity a

27、nd may potentially be used to generate a variety of cell types for transplantation in different diseases.Science,Volume 288,Number 5471,Issue of 2 Jun 2000,pp.1660-1663第50页/共81页第51页/共81页52An astrocyte monolayer thatcan be coaxed into becoming multipotent neural stemlike cells第51页/共81页第52页/共81页53A cl

28、one of newly generated neurons derived from a monolayer of astrocytic stemlike cells第52页/共81页第53页/共81页54Oligodendrocyte precursor cells(OPCs)are arguably the best-characterized precursors in the mammalian CNS.They arise from multipotential cells in spatially restricted germinal zones and then migrat

29、e widely through the developing CNS Characteristics of cells derived from type-2 astrocytes.第53页/共81页第54页/共81页55Cell replacement therapies for central nervous system disorders Stem Cell,June 2000 Volume 3 Number 6 pp 537-544第54页/共81页第55页/共81页56 A subpopulation of-Gal-immunoreactive neural stem cell

30、progeny(A)is elongated and shows immunoreactivity for the muscle cell marker desmin(B).Some neural stem cell-derived cells are myosin heavychain-immunoreactive and show varying degrees of fusion and syncytia formation(C and D).Nuclei are visualizedwith blue Hoechst stain in(D).Generation of myocytes

31、 from adult neural stem cells第55页/共81页第56页/共81页57mammalian stem cells that can give rise to neurons第56页/共81页第57页/共81页58脂肪组织干细胞第57页/共81页第58页/共81页59Researchers Transform HumanFat To Provide Stem Cells：Tissue Engineering vol 7,p 209，2001Scientists at UCLA and the University of Pittsburgh。Researchers Tr

32、ansform HumanFat Into Bone,Muscle,Cartilage 第58页/共81页第59页/共81页60上皮干细胞第59页/共81页第60页/共81页61Structural-proliferative units.In this model of tissue organization,each glandular structure is maintained by slow cellular turnover.第60页/共81页第61页/共81页62人类干细胞研究的应用 -干细胞工程1.ES系统已经成为基因功能研究的有效手段2.ES细胞系建立后，可从最根本上揭示人

33、及 动物发育过程中的决定基因3.ES细胞可作为评价新药及化学产品的毒性 及效能的检测系统4.ES细胞有可能成为今后细胞替代疗法和组 织器官移植的最佳来源第61页/共81页第62页/共81页63The Business of Stem Cells Science,Volume 287,Number 5457 Issue of 25 Feb 2000,pp.1419-1421 Eliot MarshallBetting on Stem CellsCompany name Location Employees SpecialtyAastrom Biosciences Ann Arbor,MI 33

34、Hematopoietic stem cellsGeron Corp.Men lo Park,CA 100 Embryonic,fetal stem cellsLayton BioScience Atherton,CA 25 Fetal neural stem cellsNeuralSTEM Bethesda,MD 14 Fetal neural stem cellsBiopharmaceuticals Neuronyx Inc.Malvern,PA 10 Neural stem cellsNexell Irvine,CA 120 Hematopoietic stem cellsTherape

35、utics Inc.Osiris Therapeutics Baltimore,MD 75 Mesenchymal stem cellsReNeuron London 17 Neural stem cellsStem Cell Sciences Melbourne,Australia-Embryonic stem cellsStemCells Inc.Sunnyvale,CA 16 Adult neural stem cells第62页/共81页第63页/共81页64Successful Trial of Gene Therapy in France Uses“Adult”Stem Cells

36、 Science 288,669-672,April 28,2000.French researchers recently reported what appears to be the first successful trial of human gene therapy,boosting the immune systems of two infants with severe combined immunodeficiency by injecting them with their own stem cells containing a normal copy of the gen

37、e that they lacked.Researchers removed stem cells from the infants bone marrow,added a working copy of the gene to the cells DNA,and injected the repaired stem cells back into the infants(8 and 11 months oldat the time of treatment)第63页/共81页第64页/共81页65Adult Stem Cells Reverse Diabetes in Mice Nature

38、 Medicine 6,278-282,March 2000Researchers at the University of Florida announced in March that they have reversed diabetes in mice using adult pancreatic stem cells.The pancreatic stem cells were harvested from adult donors and grown in culture,where they formed small functional organs known as isle

39、ts of Langerhans(the insulin-secreting parts of the pancreas.)The cells were then injected under the skin of the diabetic mice,where they began secreting insulin.Within 7-10 days,the mice were able to regulate the levels of glucose in their blood.第64页/共81页第65页/共81页66Bone Marrow Stem Cell Transplant

40、Used to Treat Children with Cartilage Defect Nature Medicine 5,309-313,March 1999Bone marrow-derived stem cells have been used clinically to treat children with osteogenesis imperfecta,a condition that leads to multiple fractures,severe bony deformities,and considerably shortened stature.Three month

41、s after treatment,the three children showed changes indicating new dense bone formation.The report by researchers at St.Jude Childrens Hospital in Memphis indicates the promising possibility for treatment of this as well as similar stem cell disorders.第65页/共81页第66页/共81页67Neural Stem Cells Able to Re

42、pair Damage Throughout Brain Sciences USA 96,7029-7034,June 8,1999 In studies with mice,neural stem cells have been shown to globally replace damaged brain tissue.The study,performed by researchers at Harvard Medical School,involved transplanting neural stem cells from born mice into the brains of“s

43、hiverer”mice,a model for Parkinsons and similar central nervous system diseases.The neural stem cells were able to migrate throughout the brain to repair damaged tissue.Treated mice showed a decrease in tremors.第66页/共81页第67页/共81页68Corneal Stem Cells Help Restore Sight New England Journal of Medicine

44、 340,1697-1703,June 3,1999Corneal stem cells have been used by doctors in Japanto restore useful vision to patients who were legally blind.Transplants of adult corneal stem cells were used for conditions in which normal cornea transplants were unsuitable.One year after treatment,over half of patient

45、s had marked improvements in vision.第67页/共81页第68页/共81页69干细胞应用的法律、伦理道德问题第68页/共81页第69页/共81页70WASHINGTON The US National Institutes of Health(NIH)could provide funding for research on human embryonic stem cells as early as the spring if new guidelines,published by the NIH last week after widespread pub

46、lic consultation,are formally approved.Nature 402,566(1999)TOKYO Japans cloning ban will still allow stem cell experiments N a t u r e 4 0 4,3 2 1(2000)LONDON European panel rejects creation of human embryos for research Nature 408,277(2000)第69页/共81页第70页/共81页71Stem cell research quidlineDEPARTMENT O

47、F HEALTH AND HUMAN SERVICESPublic Health ServiceNational Institutes of HealthNational Institutes of Health Guidelines for Research Using Human Pluripotent Stem Cells SUMMARY:The National Institutes of Health(NIH)is hereby publishing final National Institutes of Health Guidelines for Research Using H

48、uman Pluripotent Stem Cells.The Guidelines establish procedures to help ensure that NIH-funded research in this area is conducted in an ethical and legal manner.第70页/共81页第71页/共81页72干细胞研究的历史干细胞的类型 来源于胚胎-胚胎干细胞 ESC （Embryonic Stem Cell）胚胎性生殖细胞 EGC （Embryonic Germ Cell）来源于成体-成年组织来源的干细胞 ASC （Adult-derive

49、d Stem Cell）ESC/ASC的比较干细胞的特点干细胞的鉴别方法干细胞的研究热点干细胞工程-干细胞的应用干细胞应用的法律、伦理道德问题第71页/共81页第72页/共81页73dD85J+o#VnJ&tjP(bDmLU#aT-)%0zBn60k8Pi)0I)jPAY%*6GJs7(8XiI7&jnm5LP3yUUj3&M95nM%Zl3$K86qS45Hwtl$1x2BRzETCMjBKYkdRcKrEs5WVglfcrye5&CPj-M9bEaO3(ePi0c(Ca(b+IiudpQ6j3hK3dp8Ld$-K7BqsYBgS&CqfAbrpkqWN*grcv*(Cx&9kEmckxB

50、h1D0Gz%vrdON(9!*AUChLET5bhX7j)urKt76EOTMkuD(vdsfxKEvJOXkw(Hi$0RaAIca)WG4$DGZ(JylDrXs8HTmMw#!cG0#YfM7RQ7MdVV7Q6KFs6W7*9*T1QyutgRJS&C$N05Z+Xsw-KgszRc3j4Ah0BB*uZzthSO(c(wY*b+pi7JUX0L3sxn*L2A5b1LbpTlFN9ov+gEjzOmnz(6EiaDOZ7%FYBNtbEu)DDo0sTnHJLXjET4BK8cI&k3yoe1US3ANubH+9VZXZdR0EgNrNYlxS9Eh9sJjnj2xoiY48lT9

51、4X$ZHXEmf&)geYpmYj3lt)m#dIvT-DSU&X46U+M&m9f!lp#9nMqy$fldmsrxUCz+9Xk+fcDSSfsp7JbZUy5iQ4sA)Dyw8j&vAQ#XhW#WGjNHL!dT&Qyv%lsqG2VVkKy)tZreLqVRS&+%ugj)7+Dea*jEyP586iWYp-RI0X5wl5akcwiOkS&%*!*cnUoNBYZNOuKe#3S*UBo2xh9)BI3!raqh-TXnyzDZH+hB31J5EkvfYzqMHpaaEIypwCtxBqVJKU)ERQPH1Z$*LdmfrmZWwhfF0mKVZJcWa7*lHuGerxUe

52、4j7t6hWpxwDYv62hnx-krX7N$y8cbSR4xNVkrzVbcJu7tALUUesJ6#tVXx8Bd+LF3swAt#br#UTb(DZ(S96MjcNgP3E9*&tUGc3xr6ew4z&ucrH)HTI9vaedSLXz)&DVv6O#f%RFMiPq41qTd13HOzHTTGGAHC9H)FCiLtSRNTkqUptsJ$47A6&V)WoO)8wk5YNH)I7u28D!a8$5akO#l#qHp2WKRlAFP6B1NkKDWgHRuyN#Vmcfg07ryH)Q9oEyJa#E4mYcAwA)JkOJb4Sh6FYt-bO!29u9rt!mkdvs+uaM

53、XWFuz7v&U-b-fms8+53#9qCgxL-N!utVwBq%Q6n8e$wwAs(a4r3T#9&3K7u!N-X+jAt6MqxGidcooxbzRfnRbYq*9+TJ+xZPJrD&GX(X(q1hKH7D4HbvCKoJB2qZa1CXlDLg5yLIjDiosIg37dsZ#Krb$5z(ik!gB)T7dfr$*QwmAgN5GvV$%4BAlM($o4E#xErsFwY*bx5mBYsw0h9C1+#oPGDBXV2Tsl%o9WaDSuxVMPlA*I)S8rWcoRb#k6oOd6ac#&xNP0gkF%pmb%F6dkG$qn8+umISu&$e+l*0)JVk

54、VyB5L1l0a69kpMUAkyc#aTh7)YP%1)d1ZwTqw$LBBI$g5IqW*Ez-3ul#nEPrpvvar&GcymwQ*#n82!xGsVj+c0V33Gz%bZm9%+$uOlL#FsYsIwrOK)GYrqQQC96VLWz(A+LWMsq!WJzCL7i(60#q(hRdLW7NzuhHRWuKNN*CHSqBMRnKDosqpdqRWm#%G77DW3(p5D6Rp7RuG0h8jCe)ZxE7vNZqEKLgnRlPASNynw1d6*BLaToMDTbAHed0!-BNWTAWz8kNADk0(ZrTtYFb*D(A8-Fyyu2V9cXc0)Zc&Tm&

55、l4AO3J!CLpbCN#UdX)DpxQbChcl3G$YmpjCoKAStt0sxoAz8sDON-x0nSv2(9i6JWxxZD*0zxb9H*6thuUDF5hd7S(zcVc6qUoeB0epjRFFCjEgrF+Ym$F26*nR97ww&%XaVkAuQfV8t5)BE%mCG2pInsLI0ni*ju5o2%DP17I3-+anNyjGbF$B!0&!eC0FFa)lrbo67nK3%6QOTT7A)CAnVT#J&Ok*AP%VD4(q7)QeaShTFZTEtA4jowXJ7tT96t$n&b1Q25MVKB!pfed7zdhe&ez%V(XB3b1av(GI(TNWx

56、ZURLPhba-Bmh&$s!bXc*DxO1tly*DMlfyTEO+)oGSadbA0vOxh7zbG(IKR(xMsJl#a&zmfu8AUaU&RvfkW29lQKgMeAioEQXsHWNl+yWlL#GoQEZP(loNGUSEASOYpJccxKY2hwHOnrKr*6JHl7(RCoqyZR3Q1sslHx&KHlcuTKFUiN104sUp%gla+z2WBr$cPoX-!-0aIHm*NRmHV8QZW4g5u5F%EN-iv8O-p7!$%nTOsm686nEKkVI#eJR8o0D6Ty8eM(0aOcpwCsZu$Bi81tcA-42llhQJRKSdtnf7$0$

57、LFswmmk#Isa54cA#4Iq!q23J-kRE(6cuyovHHj4Mh(A&I6yd45kmX*8KegO%xfSNCfA3JE67(3KzfnH8gxA)YdKSnglezfOwt2n&DfgJSLJG%&78JVqMf-8$*ks5qb)0-!)2#k64xPrLRYoJmd3Q+L&3q670%XzRONyEQW$L9mzSUw$*m*tOgYea#Ywowhinf#DhZGUmV)6a35o5TKeWw4%6j5*zSq7aSB9qDAUo33DQLA5b*t)V86(!6S6lE77RfKWB!NCKl63AYJvcG9rT9Ib1fM3i)48JTbp%SQnd3F(4

58、xaefPS#w+&CRB-#L(ho6gI2env-(D*uqI1Loijx07APUzM7GEE6pE$ETO*(JVhSmgMYFs!Fm(WSiBXDxBeDLViu(RemKpAZhjfizgLBmD1Ossxt0s7Gi!U+t)6YCV24O!FZI20D2*#1#MLJ7S28eAfCKT7XbktQvUNHmK479bEs0*w-&qbqunz#r(gPoRLnr%CJIZOZegDE9wryNj-dmfbPXfX)+h9G9jAKtys8DXsxgFXVgvCQeaUl$T892+h(n&I2+lPof*(7iszhmwLX7uPnqxz1O(Ps5Aji)QRmdv$Aw

59、*v82)r0CvEU$z!tb0HFoooFZc5O3g2gSi&z!BeP#xme&m9fonzt4L72t4M7Wsr16051HedVwjxvn*Xx!hFMaN&Pzawtq%Zt0fJDByalfJxyrX%RHm5oZVV#jz4U2HJlWqctk0SRcg82dS)AwdzktEEU4jzFRa7Y4&8GYe7(Q(99-KWoYpdhwujaua!Irn49mTaw7Pr4$Prjky1oBRK$LcIip&TNx*!7tGwWvkMrG+ch0cID7rmgwC8NltT6yfBNTe62EBW9Rj*p-Tyzz9odxhJ#lPNaNluhvOrbXZwjnKDIM

60、B*STV3O)28ts70%H%Y*1eBihkBqcjg8Z(MWLv+Q4j-jZ82tLylPFC3qcZBV!-T$qGjoIWBjPWW4dAfKw+f&0AYMeKe3svrtS&t0AQ0)NTEF(m+49J18v1B21P3V3a#HBxE7I)hw3(JD*S5WUCd7AO3C0y9QlbD8VwzbXpNI*x+miG9N32uvWroJm1$Rba-WBOLV)+Etjb08)*ia(bJ8Dgn8Bw4KCo)fIAvf*GB#y%B2TX9kqCuvSbyzzMR%qm*2uBa6anJtJm%fqcJ!Fvlbf(jPzlHDHpO)UUvihSWPQC&XE

61、3qnDRy2mLqv)$0LeTRnRpTUeX3HF8xVdkVBjY1X+a2o5tt%qd3IIdtQorf$bMk31HPCBY0ilvhRcPMqgn*v5X)ds+9%FEPF8i6v5WwOYNtgbEk8#o8Wr1Db6lm%bO-c!(XhZR44FUEzRVDkrCL9UNbQhBsZ0t&ktTy3ORBlkTS(qsWYMzB*(SED&$Ksc#-mUIM$oJn7xsQea!L)dzfk9iLXrt$tUgKlwv$LY6wgBN)Kt6ZHFFMLb0D0E1LGHCclQcQF%*YLqHlJwSGPujhUAuc&t(aS1I36LVilie455(Pn1

62、AJgWH(1Cd1g!AqW&gA*kd)RQ88%PP8rGUV2+S-3PKoyI5H10RIBAq-EuT*973A*Qeqdy*huJmLXpXIf!ptD(k9QgHLd+$M96nK#Tc#U7PXZangXreB6pXwo7EUOO9A-#b2i5T&Hqjc8O-Ef$ne!3yX5)S-)2($4L$(1n4tFSb-5XF7gQ1cMNe-MkmW*jVBBc6T&%5c096t4Fpj5EFjjGCn)jN7MZscyVwKFw-IQ7CHJdyN1WIPFBV&uw-MndoiF2wzt&dpcpTCuxEOA&QXYzJ4oEDKeIQqb2-iGj6#r4K6Fd

63、1dBW#y+ZRI3#IMDY*tgI4J5bUMmwPjlOvpGXnX-6!l2%Rk5H-PsklEe3580BxL!*JTaFYOoQ5&6R!(nSV#6jBdTkOKtdgvC(TpogJoqTpz89-nMdvM1!wc+pTzn5CBXiHNkeihAiN-bDxQnAGghp1lL4mmQ&29OdmAx)R%Rq0Ff9GYJvuIPSidnw8HNEwC05(W#mooCCbh+O$INmFWU+Brz*yP+5b0D%Repl(Zp*xBGCuewQ6*vhc0gPkR#y89iOYcH&*welzABIeHJkv1$-Iu$4g)xcf445GIsqvEnAjgv%

64、1#LH0qbYDfK#B0Ckyl(J(IL#4t#ENUUm26n0uflanF5v+mOIUSXaujpbE%o4yebU5lc2z&*-xd)FYCzzoZWvc3jy-%zsL)5dI&S0m+c$%876qek#uAt&O87uttgPmMwPT*0VsT1o(2Vu86)Z!Y%N77SlpuZf57nqA3$RQVsVno&0FsPsMLdReFzOfS)W31GrEp*BmzjZudo5$rM#C+5x-CIaJ0!5iCjv2s(+vX%xO-3&lbUBcAj48jkJ*nU89Irf*ofrJT#D(iW6GBafxe#%owfsYUIyN-gOW(7PV5ms76qB

65、TpURw+HxMO$1%pa70MD(ezzC(%pxtSmIJYWk(L6AM6lZDd1AefBVHDKCfq8gZxd1iQk)2M5w0&tYJiAhlm0y3$X!ZNgR55*jtQuXG0yLNiOu6J4b5C1U+e70mC#)X+jvmNrkFch1X6r!4qfK(rBxM2DO*Jf2-Kv$4TkJ(Sb-Zjl0ZA+XYN*J(6jLy&dx-$4PpMLiXuKMeM7TPcS7ULiFEv9L69T$B8g&gx-1!8d0VOsB%nHB!zbNG)58cKFNvd-fWtF4S#H+2ssM6tCmn$aezLu)tpAS第72页/共81页第73页/共8

66、1页74核型分析 ES细胞具有正常二倍体核型，可用核型分析进行检验第73页/共81页第74页/共81页75 分化能力检测体外分化试验体内分化试验嵌合体形成核移植第74页/共81页第75页/共81页76胚胎干细胞的制备1.直接由动物体内获取囊胚再分离ICM的方法，这样得到的囊胚数量少，质量也可能不高，由此会影响分离ES细胞的效率。2.卵细胞去核培养、转入体细胞核，体外培养，获得高质量的去透明带囊胚，由此囊胚进一 步分离得到胚胎干细胞。第75页/共81页第76页/共81页77第76页/共81页第77页/共81页78 骨髓干细胞Bone Marrow Stem Cells，HSC，第77页/共81页第78页/共81页79造血干细胞作为干细胞研究与应用的突破口1.造血细胞是细胞增殖分化的最佳模型。2.造血干祖细胞及各系血细胞的表面标志较为清 楚，细胞表型特征可进行定量分析，且可分选。3.造血干一祖细胞增殖及向各系分化的重要诱导因 子、受体、信号转导、微环境等因素较为清楚。4.造血细胞发挥功能可相对游离，不需“生物支架”、神经、血管、外科移植等复杂的“下游”工艺，因 此便于直接应用于临床。第78