治疗性克隆研究的现状与展望ppt演示课件

MEDICAL CELL BIOLOGYMEDICAL CELL BIOLOGY 1 Current status and prospect of Current status and prospect of therapeutic clone studytherapeutic clone study Islet transplantation in seven patients with type 1 Islet transplantation in seven patients with type 1 diabetes mellitus using a diabetes mellitus using a glucocorticoidglucocorticoid-free -free immunosuppressive regimenimmunosuppressive regimen A.M. James Shapiro, et al.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：核心内容如下： n n 胰岛分离纯化后即刻进行移植胰岛分离纯化后即刻进行移植 n n 移植足够数量的胰岛组织（两名或以移植足够数量的胰岛组织（两名或以 上供体）上供体） n n 抗免疫排斥反应采用非类固醇激素的抗免疫排斥反应采用非类固醇激素的 药物和单克隆抗体药物和单克隆抗体 2. 胰岛移植示意图 3. Edmonton方案的最新进展 l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% l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 l胰岛移植是治愈糖尿病的潜在希望 ADA annual Meeting, 2004, Orlando 4. 胰岛移植面临的两大挑战 l胰岛组织来源：远远满足不了需要 l免疫排斥反应：移植失败的重要原因 5. Definition of Stem Cells Self-renewal Multi-lineage differentiation Cornea Pancreas Nerve Liver Skin 6. Classification of Stem Cells lDifferentiation capacity nTotipotent stem cells nPluripotent (multipotent) stem cells nOligopotent (unipotent) stem cells lSource nEmbryonic stem (ES) cells nSomatic (adult) stem cells 7. 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 8. 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 9. n1999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 里程碑里程碑 n1998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 10. Milestone nIn 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 11. 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 12. Gearhart 13. Thomson 14. Moon 15. 16. 人胚胎干细胞人胚胎干细胞(hES)(hES)的特点的特点 形态形态：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 17. n n 增殖速度增殖速度：1824h分裂增殖一次 uhES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 uhES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 n具有分化成外、中、内三个胚层的潜能 n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 18. 人胚胎干细胞的鉴定人胚胎干细胞的鉴定 l人胚胎干细胞具有正常稳定的二倍 体核型和带型 l胚胎干细胞具有较高的端粒酶活性 及碱性磷酸酶的表达 n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 19. lhES细胞有特异性表面抗原的表 达 胚胎阶阶段特异性抗 原 Stage-specific embryonic antigen SSEA-1SSEA-3SSEA-4 Thomson等分离的hES阴性弱阳性强阳性 Gearhart等分离的hES阳性弱阳性阳性 小鼠ES阳性阴性阴性 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-1SSEA-1阳性阳性可能是源于原 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 20. l具有转录因子Oct-4的表达 n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 nOct-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 21. l l 人胚胎干细胞具分化的多潜能性人胚胎干细胞具分化的多潜能性 hES 注射 小鼠皮下 混合组织瘤 Ectoderm Mesoderm Endoderm Nerve Skin Adrenal Blood Heart Muscle 22(3):265-274 www.StemC 34. 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 35. Insulin/C-peptide/DNAInsulin /Caspase-3Insulin/DNA TUNEL Insulin/DNAInsulin/FITC-Ins/DNA Artifactual insulin release from differentiated embryonic stem cells Hansson M, et al. Diabetes 2004, 53(10):2603-2609 36. Hansson M, et al. Diabetes 2004, 53(10):2603-2609 37. Key MessagesKey Messages from these two Studies lES 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 lAlthough differentiated cells contained immunoreactive insulin, they did not contain C-peptide lVariable insulin release from these cells upon glucose challenge could be found, but C-peptide release was not detected lMany of the insulin-immunoreactive cells were undergoing apoptosis or necrosis lThese 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 38. ConclusionConclusion Insulin detecting alone can overestimate genuine cell differentiation when exogenous insulin is present. Therefore, several methods should be combined for reliable analysis of insulin expression including l l C-peptide staining C-peptide staining and releasingand releasing l l Electron microscopyElectron microscopy l l Northern blotNorthern blot l l In situIn situ hybridization hybridization l l Metabolic labelingMetabolic labeling l l Demonstration Demonstration of of bi-bi- phasicphasic insulin secretion insulin secretion l l Transplantation Transplantation assays assays for for cell cell function function that that demonstrate demonstrate rescue rescue of of the the diabetic diabetic phenotype phenotype for more than a monthfor more than a month Rajagopal J, et al. Science 2003, 299(5605):