治疗性克隆研究的现状与展望ppt课件

治疗性克隆研究的现状与展望,Current status and prospect of therapeutic clone study,Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen,A.M. James Shapiro, et al. University of Alberta Hospital, Edmonton, Canada,New Engl J Med 2000，343(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,ADA annual Meeting, 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,Definition of Stem Cells,Self-renewal Multi-lineage differentiation,Cornea,Pancreas,Nerve,Liver,Skin,Classification of Stem Cells,Differentiation capacity Totipotent stem cells Pluripotent (multipotent) stem cells Oligopotent (unipotent) stem cells Source Embryonic stem (ES) cells Somatic (adult) stem cells,What is ES cells?,Primitive (undifferentiated) cells from the embryo that have the potential to become a wide variety of specialized cell types.,ES cells,Pluripotent stem cells,In vitro fertilization,Totipotent cells,Blastocyst,Inner cell mass,(ICM),-NIHs definition,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,Martin GR. PNAS, 1981, 78 (12):7634-7638 Thomson JA, et al. 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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Shamblott MJ, et al. PNAS, 1998, 95 (23):13726-13731 Gearhart JD. Science, 1998, 282 (5391):1061-1062,Milestone,In 2004, Scientists from Korea and U.S.A reported the derivation of a pluripotent embryonic stem cell line (SCNT-hES-1) from a cloned human blastocyst,Hwang WS, et al. Science 2004, 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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Shamblott MJ, et al. PNAS, 1998, 95 (23):13726-13731 Hwang WS, et al. Science 2004, 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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Hwang WS, et al. Science 2004, 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（freeder cells）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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Hwang WS, et al. Science 2004, 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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Hwang WS, et al. Science 2004, 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,Hwang WS, et al. Science 2004, 303(5664):1669-1674 Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Shamblott MJ, et al. PNAS, 1998, 95 (23):13726-13731 Thomson JA, et al. 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,Thomson JA, et al. Science, 1998, 282 (5391):1145-1147 Hwang WS, et al. Science 2004, 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,Muscle & Bone,Pancreas,Liver,Both in vivo and in vitro,彭红梅等. 北京大学学报（医学版） 2004, 36(4):431-434,碱性磷酸酶染色,SSEA-1染色,裸鼠皮下接种,腺样体结构,神经组织结构,软骨组织结构,彭红梅等. 北京大学学报（医学版） 2004, 36(6):605-608,拟胚体（EB）培养,分子标志的表达,1周,2周,3周,4周,hES 1周 2周 3周 4周 MEF,Lanes,Oct-4,Nestin,NSE,GFAP,SNAP,NFH,PDX-1,Insulin,Glucagon,AFP,Globulin,GAPDH,MEF = mouse embryonic fibroblasts NSE = neuron specific enolase GFAP= glial fibrillary acidic protein SNAP= synaptosome-associated protein NFH = neurofilament heavy chain,From Embryonic Stem Cells to Insulin-Secreting Cells,胚胎干细胞分化为胰岛细胞的路径,ES cells,Ectoderm,From mouse ES cells to insulin-secreting pancreatic islet cluster (Five-stage protocol),Lumelsky N, et al. Science 2001, 292(5520):1389-1394,Stage 1 Expansion of ES cells,Stage 2 Generation of EBs,Stage 3 Selection of nestincells,Stage 4 Expansion of pancreatic endocrine progenitor cells,Stage 5 Induction of differentiation and morphogenesis of insulin-secreting islet cluster,2-3 days,4 days,6-7 days,6 days,6 days,Lumelsky N, et al. Science 2001, 292(5520):1389-1394,Lumelsky N, et al. Science 2001, 292(5520):1389-1394,主要结论,从小鼠ES细胞诱导分化产生可表达胰岛素和其它胰腺内分泌激素的细胞 该细胞可自聚集形成类似正常胰岛的三维结构 葡萄糖可促发这些细胞群释放胰岛素，其机制类似体内的刺激作用 移植到糖尿病小鼠体内时，这些胰岛素产生细胞出现快速的血管化，并保持胰岛样结构，但没有明显的降血糖作用,Lumelsky N, et al. Science 2001, 292(5520):1389-1394,From human ES cells to insulin-secreting cells,Schuldiner M, et al. PNAS 2000, 97(21):11307-11312 Assady S, et al. Diabetes 2001, 50(8):1691-1697 Segev H, et al. Stem Cells 2004, 22(3):265-274,5 day,10 day,Further induction,?,Assady S, et al. Diabetes 2001, 50(8):1691-1697,Insulin,GK,Glut-2,Glut-1,Oct-4,Ngn-3,PDX-1,b-actin,Differentiation (days),0,7,15,22,28,10,30,uhES,EB,dhES,NHF,NC,Assady S, et al. Diabetes 2001, 50(8):1691-1697,NHF = normal human fibroblasts,Differentiation of Human Embryonic Stem Cells into Insulin-Producing Clusters,HANNA SEGEV, BETTINA FISHMAN, ANNA ZISKIND, MARGARITA SHULMAN, JOSEPH ITSKOVITZ-ELDOR,STEM CELLS 2004;22(3):265-274 www.StemC,Insulin staining,TUNEL+ nuclei,DAPI stain for nuclei,Merge (A-C),Insulin staining,FITC-Insulin uptake,Insulin staining of ES cell progeny from insulin uptake Rajagopal J, et al. Science 2003, 299(5605):363,Insulin/C-peptide/DNA,Insulin /Caspase-3,Insulin/DNA,TUNEL,Insulin/DNA,Insulin/FITC-Ins/DNA,Artifactual insulin release from differentiated embryonic stem cells Hansson M, et al. Diabetes 2004, 53(10):2603-2609,Hansson M, et al. Diabetes 2004, 53(10):2603-2609,Key Messages from these two Studies,ES cells differentiated in media without exogenous insulin did not stain for insulin, and differentiated ES cells subsequently cultured in insulin-deficient media lost insulin staining Although differentiated cells contained immunoreactive insulin, they did not contain C-peptide Variable insulin release from these cells upon glucose challenge could be found, but C-peptide release was not detected Many of the insulin-immunoreactive cells were undergoing apoptosis or necrosis These cells took up fluorescein isothiocyanate (FITC)-labeled insulin from the culture medium Suggesting that containing insulin in these cells is not as a result of biosynthesis but from the uptake of exogenous insulin,Rajagopal J, et al. Science 2003, 299(5605):363 Hansson M, et al. Diabetes 2004, 53(10):2603-2609,Conclusion,Insulin detecting alone can overestimate genuine b cell differentiation when exogenous insulin is present. Therefore, several methods should be combined for reliable analysis of insulin expression including,C-peptide staining and releasing Electron microscopy Northern blot In situ hybridization Metabolic labeling,Demonstration of bi-phasic insulin secret