人骨髓来源的多能成体祖细胞在体外定向诱导分化为肝样细胞的实验研究
|
摘要
肝炎、肝硬化及肝癌等肝脏疾病所至的终末期肝功能障碍，目前最有效的治疗手段是原位肝移植(OLT)。然而，由于供体的缺乏、免疫排斥和高额费用等问题，OLT的广泛应用受到了限制。杂交型生物人工肝(HBAL)的目的是使终末期肝病病人桥接(bridge)肝移植，而且肝细胞具有强大的再生能力，临时应用生物人工肝也可以解决急性肝功能的失代偿，促进肝细胞再生，部分患者肝功能完全自然恢复，病情康复。但由于作为HBAL核心生物材料的肝细胞来源问题未得到根本解决，使HBAL的临床应用远未达到理论上的预期效果。研究证明人骨髓来源的多能成体祖细胞(MAPCs)具有向肝样细胞分化的潜能，由于其具有易于调控、供源丰富、容易获取、有自体供源、避免免疫排斥等优点，因此，有望成为理想的肝组织工程细胞来源。
第一部分：人骨髓来源的多能成体祖细胞分离纯化及体外培养的实验研究
目的 建立体外分选纯化人MAPCs的方法，用自行配制的细胞培养基对MAPCs进行体外培养，了解其生长、增殖特性。
方法 取志愿者适量骨髓后采用梯度密度离心法分离获取单个核细胞，对单个核细胞行贴壁培养后，将获取的骨髓贴壁细胞通过CD45、GlyA免疫微磁珠负分选，流式细胞仪鉴定分选后细胞纯度；培养观察细胞生长情况，对传代到不同阶段的细胞进行CD45、GlyA流式细胞分析，初步了解MAPCs在培养、增殖过程中的免疫学稳定性。
结果 (1)通过MACS分选，平均每1×10~6／ml骨髓贴壁细胞可分选出约5—10×10~4／ml数量级的MAPCs，分选前后细胞活力分别为(96.7±1.7)％和(96.0±2.4)％，无明显差异；(2)用我们自制的
培养基培养分选后的MAPcs，细胞生长良好，最长传代到第16代;(3)
流式细胞仪分析获取的CD45一、GlyA一细胞纯度大于98%;流式细胞仪
分析传代第4、8、12代MA卫Cs，eD45一、GlyA一细胞纯度大于95%。
结论(1) CD45、GlyA免疫微磁珠负分选可从骨髓中分离高纯度
的M妙cs;(2)MAPcs在体外有较强的增殖能力，且能保持较长时间
的稳定状态;(3)我们自行研制的人MAPCs培养基能成功体外培养
MAPCS。
第二部分:人骨髓来源的多能成体祖细胞在不同诱导条件下向肝样细胞
分化的实验研究
目的探索人骨髓来源MAPCs在不同条件下诱导分化为肝细胞的
可行性;摸索并确立骨髓MAPCs诱导为肝细胞的适当培养方法及条件，
建立一个获得“骨髓源MAPCs”的成熟技术平台，为其在组织工程学研
究和临床医学中的广泛应用奠定基础。
方法(一)分组:A组:H邵(Zong/ml)、B组:FGF一4(10ng/ml)、C
组:HGF(Zong/ml)+FGF一4(long/ml)、D组:24小时70%肝叶切除兔门
静脉血清(RPVS，10%浓度)诱导分化MAPCs，E组(阳性对照组):
L一02人肝细胞株、F组(阴性对照组):未加任何诱导因素的MAPCs;
(2)免疫细胞化学、免疫荧光化学鉴定各组不同诱导分化阶段细胞的
ALB、AFP、CK一18、CK一19等肝细胞特征的表型变化并计数阳性细胞比
率;(3)RT一 CR检测A、B、C组不同诱导分化阶段细胞的ALB、AFP、
e玲18、eK一19的mRNA转录;(4)westem blot检测B、e组诱导分化
第21天、35天后细胞的ALB的表达。
结果(l)免疫细胞化学、免疫荧光化学结果:ALB、CK-18在A、
B、c、D、E组中基本为阳性着色;cK一19均为阴性着色，仅D组有阳
性着色;AFP作为早期不成熟肝细胞的一个标志，在A、B、C组的诱导
早期基本为阳性着色，在诱导中、晚期基本为阴性着色，仅D组在诱导
中、晚期有阳性着色。计数阳性细胞比率显示:HGF及FGF一4均能诱导
MAPcs向肝样细胞分化，HGF+FGF一4诱导强于二种细胞因子单独诱导，
我们自行制作的即VS诱导分化能力最强，各种肝细胞表型标志表达的
阳性率最高;(2)RT一CR结果:作为不成熟肝细胞表型的AFP，在诱导
分化的第7天，A、B、C组mRNA均有阳性表达，CK-19在分化的第
21天mRNA均为阴性;作为成熟肝细胞表型的ALB及CK一18，各组在
不同时间段mRNA的表达基本都为阳性;(3) Westemblot检测结果:B、
C组在诱导分化第21天、35(38)天，ALB均有阳性表达。
结论(l)人骨髓来源的MAPCs具有向肝样细胞分化的潜能;(2)
HGF及FGF一4具有诱导人骨髓来源的MAPCs向肝样细胞分化的能力，
HGF+FGF一4诱导强于二种细胞因子单独诱导;(3)24小时70%肝叶切
除兔门静脉血清(对Vs)具有诱导人骨髓来源的MAPCs向肝样细胞分
化的能力，且强于细胞因子诱导。
The treatment of liver diseases, such as hepatitis, liver cirrhosis and liver cancer, is always a global difficulty. To end-stage liver failture led by these disorders, orthotopic liver transplantation(OLT) is the only established treatment, but it is associated with donor shortage, immune rejection, and high cost, which are the limitations of this therapy. The treatment purpose of Hybrid bioartificial liver(HBAL) to the end-stage liver failure patient is bridged for OLT. On the other hand, the function of hepatocyte repopulation is mighty, applied HBAL temporarily may obtain some purpose of treating anxious liver function, accelerating hepatocyte repopulation, supporting liver function renenw, and healing the state of an illness. However, the anticipated clinical efficacy of HBAL has not proved yet, mainly because of the source deficiency of hepatocytes which are the core bioartificial materials of HBAL.The investigation have proved that multipotent adult progenitor cells(MAPCs) from human bone marrow can d
ifferentiate into functional hepatocyte-like cells. MAPCs may be a relative ideal cells for liver tissue engineering as they are more easily to be manipulated, have the merits of abundant donor source and easy to harvest, and be obtainted autologously to avoid immune rejection. Part I : The investigations on purification and culture of multipotent
adult progenitor cells from human bone marrow Objective To create a method of separating human MAPCs in vitro using immunomagnetic beads. To investigate the possibilities of MAPCs
-5-
producting and proliferating by cell culture produced by ourselves.
Methods The bone marrow mononuclear cells were obtained by inspiring the proper volume of bone marrow from volunteers and then centrifuging by gradient and density. The mononuclear cells were cultivated by adherent culture. Collected the bone marrow adherent cells, and isolated the adherent cells by MACS through depletion selection by use of CD45 and GlyA microbeads. Analyzing the component of the CD45~>. GlyA" cells by flow cytometers. Observed the cells' form hi different growing phase. Flow cytometers analyzed the subculture cells at different time point in order to investigate the immunization stability of MAPCs producting and proliferating by cell culture.
Results (1) Per lX106/ml bone marrow mononuclear cells could separated 5 ?10X104/ml MAPCs by MACS. The activity of the pro-purification or after purification cells was (96.7?.7) %and (96.0?.4) % respectively, which was not different significantly with that before purification. (2) The MAPCs grew well in the cell cultures produced by ourselves. We observed that MAPCs could be expanded for more than 16 population doubling. (3) The purity of the CD45~% GlyA~cells separated from bone marrow adherent cells was more than 98 % by flow cytometers. The purity of MAPCs which had cultivated 4, 8 and 12 cell doublings were more than 98 % by flow cytometers.
Conclusions (1) The MAPCs derived from bone marrow can be purified by MACS through depletion selection by use of CD45 and GlyA microbeads, and they can survive and differentiate after tune. (2) The abilities of MAPCs producting and proliferating by cell cultures is active, and can keep non-differentiation state for a long time. (3) MAPCs cultures produced by ourselves is appropriate MAPCs culture in vitro.
Part II: The investigations of multipotent adult progenitor cells from
human bone marrow differentiating into hepatocyte-like cells in the
different appropriate condition in vitro
Objective To investigate the possibility of the MAPCs from human
-6-
bone marrow which are induced into hepatocytes in vitro. To explore the feasibility of differentiation from the MAPCs into the hepatocytes, and to establish the method and condition of MAPCs culture in vitro. And then, to make the hepatocytes derived from MAPCs as germ cell to be useable in tissue-engineering and clinical medical.
Methods (l)Divided groupsrA: Hepatocyte growth factor (HGF, 20ng/ml), B: Fibroblast growth factor-4(FGF-4 , 20ng/ml),D: HGF(20n
第一部分：人骨髓来源的多能成体祖细胞分离纯化及体外培养的实验研究
目的 建立体外分选纯化人MAPCs的方法，用自行配制的细胞培养基对MAPCs进行体外培养，了解其生长、增殖特性。
方法 取志愿者适量骨髓后采用梯度密度离心法分离获取单个核细胞，对单个核细胞行贴壁培养后，将获取的骨髓贴壁细胞通过CD45、GlyA免疫微磁珠负分选，流式细胞仪鉴定分选后细胞纯度；培养观察细胞生长情况，对传代到不同阶段的细胞进行CD45、GlyA流式细胞分析，初步了解MAPCs在培养、增殖过程中的免疫学稳定性。
结果 (1)通过MACS分选，平均每1×10~6／ml骨髓贴壁细胞可分选出约5—10×10~4／ml数量级的MAPCs，分选前后细胞活力分别为(96.7±1.7)％和(96.0±2.4)％，无明显差异；(2)用我们自制的
培养基培养分选后的MAPcs，细胞生长良好，最长传代到第16代;(3)
流式细胞仪分析获取的CD45一、GlyA一细胞纯度大于98%;流式细胞仪
分析传代第4、8、12代MA卫Cs，eD45一、GlyA一细胞纯度大于95%。
结论(1) CD45、GlyA免疫微磁珠负分选可从骨髓中分离高纯度
的M妙cs;(2)MAPcs在体外有较强的增殖能力，且能保持较长时间
的稳定状态;(3)我们自行研制的人MAPCs培养基能成功体外培养
MAPCS。
第二部分:人骨髓来源的多能成体祖细胞在不同诱导条件下向肝样细胞
分化的实验研究
目的探索人骨髓来源MAPCs在不同条件下诱导分化为肝细胞的
可行性;摸索并确立骨髓MAPCs诱导为肝细胞的适当培养方法及条件，
建立一个获得“骨髓源MAPCs”的成熟技术平台，为其在组织工程学研
究和临床医学中的广泛应用奠定基础。
方法(一)分组:A组:H邵(Zong/ml)、B组:FGF一4(10ng/ml)、C
组:HGF(Zong/ml)+FGF一4(long/ml)、D组:24小时70%肝叶切除兔门
静脉血清(RPVS，10%浓度)诱导分化MAPCs，E组(阳性对照组):
L一02人肝细胞株、F组(阴性对照组):未加任何诱导因素的MAPCs;
(2)免疫细胞化学、免疫荧光化学鉴定各组不同诱导分化阶段细胞的
ALB、AFP、CK一18、CK一19等肝细胞特征的表型变化并计数阳性细胞比
率;(3)RT一 CR检测A、B、C组不同诱导分化阶段细胞的ALB、AFP、
e玲18、eK一19的mRNA转录;(4)westem blot检测B、e组诱导分化
第21天、35天后细胞的ALB的表达。
结果(l)免疫细胞化学、免疫荧光化学结果:ALB、CK-18在A、
B、c、D、E组中基本为阳性着色;cK一19均为阴性着色，仅D组有阳
性着色;AFP作为早期不成熟肝细胞的一个标志，在A、B、C组的诱导
早期基本为阳性着色，在诱导中、晚期基本为阴性着色，仅D组在诱导
中、晚期有阳性着色。计数阳性细胞比率显示:HGF及FGF一4均能诱导
MAPcs向肝样细胞分化，HGF+FGF一4诱导强于二种细胞因子单独诱导，
我们自行制作的即VS诱导分化能力最强，各种肝细胞表型标志表达的
阳性率最高;(2)RT一CR结果:作为不成熟肝细胞表型的AFP，在诱导
分化的第7天，A、B、C组mRNA均有阳性表达，CK-19在分化的第
21天mRNA均为阴性;作为成熟肝细胞表型的ALB及CK一18，各组在
不同时间段mRNA的表达基本都为阳性;(3) Westemblot检测结果:B、
C组在诱导分化第21天、35(38)天，ALB均有阳性表达。
结论(l)人骨髓来源的MAPCs具有向肝样细胞分化的潜能;(2)
HGF及FGF一4具有诱导人骨髓来源的MAPCs向肝样细胞分化的能力，
HGF+FGF一4诱导强于二种细胞因子单独诱导;(3)24小时70%肝叶切
除兔门静脉血清(对Vs)具有诱导人骨髓来源的MAPCs向肝样细胞分
化的能力，且强于细胞因子诱导。
The treatment of liver diseases, such as hepatitis, liver cirrhosis and liver cancer, is always a global difficulty. To end-stage liver failture led by these disorders, orthotopic liver transplantation(OLT) is the only established treatment, but it is associated with donor shortage, immune rejection, and high cost, which are the limitations of this therapy. The treatment purpose of Hybrid bioartificial liver(HBAL) to the end-stage liver failure patient is bridged for OLT. On the other hand, the function of hepatocyte repopulation is mighty, applied HBAL temporarily may obtain some purpose of treating anxious liver function, accelerating hepatocyte repopulation, supporting liver function renenw, and healing the state of an illness. However, the anticipated clinical efficacy of HBAL has not proved yet, mainly because of the source deficiency of hepatocytes which are the core bioartificial materials of HBAL.The investigation have proved that multipotent adult progenitor cells(MAPCs) from human bone marrow can d
ifferentiate into functional hepatocyte-like cells. MAPCs may be a relative ideal cells for liver tissue engineering as they are more easily to be manipulated, have the merits of abundant donor source and easy to harvest, and be obtainted autologously to avoid immune rejection. Part I : The investigations on purification and culture of multipotent
adult progenitor cells from human bone marrow Objective To create a method of separating human MAPCs in vitro using immunomagnetic beads. To investigate the possibilities of MAPCs
-5-
producting and proliferating by cell culture produced by ourselves.
Methods The bone marrow mononuclear cells were obtained by inspiring the proper volume of bone marrow from volunteers and then centrifuging by gradient and density. The mononuclear cells were cultivated by adherent culture. Collected the bone marrow adherent cells, and isolated the adherent cells by MACS through depletion selection by use of CD45 and GlyA microbeads. Analyzing the component of the CD45~>. GlyA" cells by flow cytometers. Observed the cells' form hi different growing phase. Flow cytometers analyzed the subculture cells at different time point in order to investigate the immunization stability of MAPCs producting and proliferating by cell culture.
Results (1) Per lX106/ml bone marrow mononuclear cells could separated 5 ?10X104/ml MAPCs by MACS. The activity of the pro-purification or after purification cells was (96.7?.7) %and (96.0?.4) % respectively, which was not different significantly with that before purification. (2) The MAPCs grew well in the cell cultures produced by ourselves. We observed that MAPCs could be expanded for more than 16 population doubling. (3) The purity of the CD45~% GlyA~cells separated from bone marrow adherent cells was more than 98 % by flow cytometers. The purity of MAPCs which had cultivated 4, 8 and 12 cell doublings were more than 98 % by flow cytometers.
Conclusions (1) The MAPCs derived from bone marrow can be purified by MACS through depletion selection by use of CD45 and GlyA microbeads, and they can survive and differentiate after tune. (2) The abilities of MAPCs producting and proliferating by cell cultures is active, and can keep non-differentiation state for a long time. (3) MAPCs cultures produced by ourselves is appropriate MAPCs culture in vitro.
Part II: The investigations of multipotent adult progenitor cells from
human bone marrow differentiating into hepatocyte-like cells in the
different appropriate condition in vitro
Objective To investigate the possibility of the MAPCs from human
-6-
bone marrow which are induced into hepatocytes in vitro. To explore the feasibility of differentiation from the MAPCs into the hepatocytes, and to establish the method and condition of MAPCs culture in vitro. And then, to make the hepatocytes derived from MAPCs as germ cell to be useable in tissue-engineering and clinical medical.
Methods (l)Divided groupsrA: Hepatocyte growth factor (HGF, 20ng/ml), B: Fibroblast growth factor-4(FGF-4 , 20ng/ml),D: HGF(20n
引文
1. Goodrich EO, Jr. Welch HF, Nelson J A, et al. Homotransplantation of the canine liver. Surgery.1956,39:244-251
2. Starzl TE, Marchirro TL, von Kaukla KN, et al. Homotransplantation of the liver in humans. Surg Gynec Obstet.1963,117:659-676
3. National Institute of Health Consensus Development Conference Statement; Liver Transpplantion June 20-23. Hepatology,.1983,4: 107-110
4. Funattsu K,Ijima H,Nakazawa K, et al. Hybrid artificial liver using hepatocyte organoid culture. Artif Organs. 2001,25(3):194-200
5. Bismuth H, Houssin D. Reduced-size orthotopic liver graft in hepatic transplantation in children. Surgery. 1984, 95:367-370
6. Allen JW,Hassanein T,Bhatia S. Advances in bioartificial liver devices. Hepatology. 2001,34(3):447-454
7. Cuervas MV, Colas A,Rivera JA,et al. In vivo efficacy of a bioartificial liver in improving spontaneous recovery from fulminant hepatic failure: a controlled study in pigs. Transplantion. 2000,69(3):337-344
8. Hughes RD,Williams R. Use of artificial and bioartificial liver support devices. Semin.Liver Dis. 1996,16:435-444
9. Jauregui HO,Naik S,Santangini H,et al. The use of microcarrier-roller bottle culture for large-scale production of porcine hepatocytes. Tissue Eng. 1997,3:17-25
10. Sielaff TD,Hu MY,Rao S,et al. A technique for porcine hepatocyte harvest and description of differentiated functions in static culture. Transplantation. 1995,59:1459-1463
11. Jauregui HO,Trenkler DM,Naik S,et al. In vivo evaluation of a hollow fibre liver assist device. Hepatology. 1995,21:460-469
12. Watanabe FD,Mullon CJ,Hewitt WR, et al. Clinical experience with a bioartificial liver in the treatment of severe liver failure. A phase Ⅰ clinical trial. Ann Surg. 1997,225:484-494
13. Nagamori S,Hasumura S,Matsuura,T,et al. Developments in bioartificial liver research:concepts,performance and application. Gastroenterology. 2000,35:493-503
14. Wood RP,Katz SM,Ozaki CF,et al. Extracorporeal liver assist device(ELAD):a preliminary report. Transplant Proc. 1993,25:53-54
15. Weiss RA. The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease. Philos Trans R Soc Lond B Biol Sci. 2001, 356(1410):957-77
16. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1998,78:7634-7638
17. Thomson JA,Itskovitz-Eldor J, Shapiro SS,et al. Embryonic stem cell lines derived from human blastocysis. Science. 1998,282:114-117
18. Shamblott M,Axelman J,WANG S,et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A.1998,95:13726-13731
19. Reubinoff BE,Pera MF,Fong CY,Trounson A,Bongso A. Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro. Nat Biotechnol. 2000,18:399-404
20. Frankel MS. In search of stem cell policy. Science. 2000,287:1397
21. Ruth Kirschstein,Lana R.Skirboll 主编.Stem Cell:Scientific Progress and Future Research Direction. Report Prepared by The National Institutes of Health. 2001
22. Stuart Forbes,Pamela Vig,Howard Thomas,et al. Hepatic stem cell. J Pathology. 2002,197:510-518
23. Theise ND,Saxena R, Portmann BC,et al. The canal of Hering and hepatic stem cells in humans. Hepatology. 1999,30:1425-1433
24. Thorgerirsson SS. Hepatic stem cells in liver regeneration. Faseb J. 1996,10:1249-1256
25. Badve S,Logdberg L,Sokhi R, et al. An antigen reacting with daslmonoclonal antibody is ontogenically regulated in diverse organs
including liver and indicates sharing of developmental mechanisms among cell lineages. Pathobiology. 2000,68:76-78
26. Stanworth SJ,Mewland AC. Stem cell: progress in research and edging towards the clinical setting. Clin Med. 2001,1:378-382
27. Goodell MA,Jackson KA,Majka SM,et al. Stem cell plasticity in muscle and bone marrow. Ann NY Acad Sci. 2001,938:208-218
28. Almeida-Porada G,Porada C,Zanjani ED. Adult stem cell plasticity and methods of detection. Rev Clin Exp Hematol. 2001,5:26-41
29. Bema G, Leon-Quinto T,Ensenat-Waser R, et al. Stem cells and diabetes. Biomed Pharmacother. 2001,55:206-212
30. Rao MS,Mattson MP. Stem cells and aging: expanding the possibilities. Mech Aging Dev. 2001,122:713-734
31. Grompe M. The role of bone marrow stem cells in liver regeneration. Semin Liver Dis. 2003,23(4):363-72.
32. Petersen BE, Bowen WC, Patrene KD, et ai. Bone marrow as a potential source of hepatic oval cells. Science 1999,284:1168-1170
33. Zhan YT, Wei L, Chen HS, et al. Differentiation of bone marrow stem cells in rat hepatic fibrogenesis environment. Zhonghua Gan Zang Bing Za Zhi. 2003,11(11):673-5.
34. Dahlke MH, Popp FC, Bahlmann FH, et al. Liver regeneration in a retrorsine/CC14-induced acute liver failure model: do bone marrow-derived cells contribute? J Hepatol. 2003,39(3):365-73.
35. Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology. 2000,31:235-240
36. Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells. Nature. 2000,406:257
37. Theise ND, Nimmakayalu M, Gardner R, et al. Liver from bone marrow in humans. Hepatology. 2000,32:11-16
38. Osawa M, Hanada K, Hamada H, et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Science. 1996,273:242-245
39. Satdarshan P.S.Monga, Yi Tang, Fabio Candotti, et al. Expansion of hepatic and hematopoietic stem cell utilizing mouse embryonic liver explant. Cell Transplantation. 2001,10:81-89
40. Terai S, Sakaida I, Yamamoto N, et al. An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes. J Biochem (Tokyo). 2003,134(4):551-8.
41. Pittenger MF,et al. Multilineage potential of adult human mesenchymal stem cell. Science. 1999,284:143-147
42. Morayma Reyes, M,roy Lund,Catherine M.Verfaile et al. Purification and ex vivo expansion of postnatal human marrow mesodermal rogenitor cells. Blood 2001,98:2615-2625
43. Morayma Reyes,Arkadiusz Dudek, Catherine M. Verfaillie,et al. Origin of endothelial progenitors in human post-natal bone marrow. J.Clin Invest. 2002,109:337-346
44.Robert E.Schwartz,Morayma Reyes,Catherine M.Verfailie,et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest. 2002,109:1291-1302
45.Yuehua Jiang,Balkrishna N.Jahagirdar, Catherine M.Verfailie,et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002,418(6893):41-49
46.Yuehua Jiang, Ben Vaessen, Catherine M.Verfailie, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow,muscle,and brain. Exp Hematol. 2002, 30:896-904
47.Morayma Reyes, Catherine M.Verfailie. Characterization of Multipotent adult progenitor cells, a subpopulation of mesenchymal stem ceils. Ann. NY Acad. Sci. 2001,938:231-235
48.Wagers AJ, Sherwood RI, Christensen JL, et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002, 297(5590):2256-2259.
49. Friedenstein A J. Precursor cells of mechanocytes. Int Rev
Cytol. 1976,47:327-355
50. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subculativation and following cryopreservation Cell Biochem. 1997,64(2):278.
51. Pot Phillips. New surgical approaches to Parkonson disease. 美国医学杂质中文版. 2000,19(2):96-97
52. Wynter EA, Coutinho LH, Marsh JC,et al. Comparison of purity and enrichment of CD34+ cell of bone marrow, umbilical cord and peripheral blood (primed of apheresis) using five separation systems. Stem Cells. 1995,13:524-532
53. Pittenger M F,Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999,284 (5411): 143-147.
54. Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA. 2001,98(14): 7841-7845.
55．张春雨，段恩奎，曾国庆．白血病抑制因子及其在胚胎发育和胚泡着床中的生理功能．生理学进展．1997，28：240-242
56.Yamamoto N, Terai S, Ohata S,et al.A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver.Biochem Biophys Res Commun. 2004, 313(4):1110-1118.
57. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000,6:1229-1234
58. Oh SH, Miyazaki M, Kouchi H, et al. Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun. 2000,279(2):500-504
59. Okumoto K, Saito T, Hattori E,et al.Differentiation of bone marrow cells
into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation. Biochem Biophys Res Commun. 2003,304(4):691-5.
60. Michalopoulos GK, DeFrances MC. Liver regeneration. Science. 1997; 276 (5309):60-6
61.Weng YS, Lin HY, Hsiang YJ, et al.The effects of different growth factors on human bone marrow stromal cells differentiating into hepatocyte-like eells.Adv Exp Med Biol. 2003,534:119-28
62. Edith Arany, David J.Hill. Ontogeny of fibroblast growth factor in the early development of the rat endocrine pancreas. Pediatric Res. 2000, 48:389-403
63. Koh K, Ohta K, Nakamae H, et al. Differential effects of fibroblast growth factor-4, epidermal growth factor and transforming growth factor-betal on functional development of stromal layers in acute myeloid leukemia. Leuk Res. 2002,26:933
64. Leffert HL, Koch K S, Moran T. Hormonal control of rat liver regeneration. Gastroenterology. 1979,76:1470-1479
65．唐望先，张国，王俊平等．流式细胞术动态检测肝大部分切除后再生相关因子的实验研究．同济医科大学学报．2001，30：330-332
66．李玉昌，马军，林俊堂等．大鼠短间隔连续部分肝切除后ADAMs mRNA表达分析．胃肠病学和肝病学杂志．2001，10：235-237
67. Stone A L, Kroeger M, Sang Q X A, et al. Structure-function analysis of the ADAM family of disintegrin-like and metalloproteinase-containing protein. J protein chen. 1999, 18(4):447-465
68. Grompe M, Laconi E, Shatritz DA. Principles of therapeutic liver repopulation. Semin Liver Dis. 1999,19:7-14
69. Laconi S, Curreli F, Dianna S, et al. Liver regeneration in response to partial hepatectomy in rats treated with retrorsine:a kinetic study. J Hepatol. 1999, 31:1069-1074
70．陈柯，高毅，潘玉仙等．有效原代猪肝细胞培养体系的研制．世界华人消化杂志．1999，7(3)：206-209
2. Starzl TE, Marchirro TL, von Kaukla KN, et al. Homotransplantation of the liver in humans. Surg Gynec Obstet.1963,117:659-676
3. National Institute of Health Consensus Development Conference Statement; Liver Transpplantion June 20-23. Hepatology,.1983,4: 107-110
4. Funattsu K,Ijima H,Nakazawa K, et al. Hybrid artificial liver using hepatocyte organoid culture. Artif Organs. 2001,25(3):194-200
5. Bismuth H, Houssin D. Reduced-size orthotopic liver graft in hepatic transplantation in children. Surgery. 1984, 95:367-370
6. Allen JW,Hassanein T,Bhatia S. Advances in bioartificial liver devices. Hepatology. 2001,34(3):447-454
7. Cuervas MV, Colas A,Rivera JA,et al. In vivo efficacy of a bioartificial liver in improving spontaneous recovery from fulminant hepatic failure: a controlled study in pigs. Transplantion. 2000,69(3):337-344
8. Hughes RD,Williams R. Use of artificial and bioartificial liver support devices. Semin.Liver Dis. 1996,16:435-444
9. Jauregui HO,Naik S,Santangini H,et al. The use of microcarrier-roller bottle culture for large-scale production of porcine hepatocytes. Tissue Eng. 1997,3:17-25
10. Sielaff TD,Hu MY,Rao S,et al. A technique for porcine hepatocyte harvest and description of differentiated functions in static culture. Transplantation. 1995,59:1459-1463
11. Jauregui HO,Trenkler DM,Naik S,et al. In vivo evaluation of a hollow fibre liver assist device. Hepatology. 1995,21:460-469
12. Watanabe FD,Mullon CJ,Hewitt WR, et al. Clinical experience with a bioartificial liver in the treatment of severe liver failure. A phase Ⅰ clinical trial. Ann Surg. 1997,225:484-494
13. Nagamori S,Hasumura S,Matsuura,T,et al. Developments in bioartificial liver research:concepts,performance and application. Gastroenterology. 2000,35:493-503
14. Wood RP,Katz SM,Ozaki CF,et al. Extracorporeal liver assist device(ELAD):a preliminary report. Transplant Proc. 1993,25:53-54
15. Weiss RA. The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease. Philos Trans R Soc Lond B Biol Sci. 2001, 356(1410):957-77
16. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1998,78:7634-7638
17. Thomson JA,Itskovitz-Eldor J, Shapiro SS,et al. Embryonic stem cell lines derived from human blastocysis. Science. 1998,282:114-117
18. Shamblott M,Axelman J,WANG S,et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A.1998,95:13726-13731
19. Reubinoff BE,Pera MF,Fong CY,Trounson A,Bongso A. Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro. Nat Biotechnol. 2000,18:399-404
20. Frankel MS. In search of stem cell policy. Science. 2000,287:1397
21. Ruth Kirschstein,Lana R.Skirboll 主编.Stem Cell:Scientific Progress and Future Research Direction. Report Prepared by The National Institutes of Health. 2001
22. Stuart Forbes,Pamela Vig,Howard Thomas,et al. Hepatic stem cell. J Pathology. 2002,197:510-518
23. Theise ND,Saxena R, Portmann BC,et al. The canal of Hering and hepatic stem cells in humans. Hepatology. 1999,30:1425-1433
24. Thorgerirsson SS. Hepatic stem cells in liver regeneration. Faseb J. 1996,10:1249-1256
25. Badve S,Logdberg L,Sokhi R, et al. An antigen reacting with daslmonoclonal antibody is ontogenically regulated in diverse organs
including liver and indicates sharing of developmental mechanisms among cell lineages. Pathobiology. 2000,68:76-78
26. Stanworth SJ,Mewland AC. Stem cell: progress in research and edging towards the clinical setting. Clin Med. 2001,1:378-382
27. Goodell MA,Jackson KA,Majka SM,et al. Stem cell plasticity in muscle and bone marrow. Ann NY Acad Sci. 2001,938:208-218
28. Almeida-Porada G,Porada C,Zanjani ED. Adult stem cell plasticity and methods of detection. Rev Clin Exp Hematol. 2001,5:26-41
29. Bema G, Leon-Quinto T,Ensenat-Waser R, et al. Stem cells and diabetes. Biomed Pharmacother. 2001,55:206-212
30. Rao MS,Mattson MP. Stem cells and aging: expanding the possibilities. Mech Aging Dev. 2001,122:713-734
31. Grompe M. The role of bone marrow stem cells in liver regeneration. Semin Liver Dis. 2003,23(4):363-72.
32. Petersen BE, Bowen WC, Patrene KD, et ai. Bone marrow as a potential source of hepatic oval cells. Science 1999,284:1168-1170
33. Zhan YT, Wei L, Chen HS, et al. Differentiation of bone marrow stem cells in rat hepatic fibrogenesis environment. Zhonghua Gan Zang Bing Za Zhi. 2003,11(11):673-5.
34. Dahlke MH, Popp FC, Bahlmann FH, et al. Liver regeneration in a retrorsine/CC14-induced acute liver failure model: do bone marrow-derived cells contribute? J Hepatol. 2003,39(3):365-73.
35. Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology. 2000,31:235-240
36. Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells. Nature. 2000,406:257
37. Theise ND, Nimmakayalu M, Gardner R, et al. Liver from bone marrow in humans. Hepatology. 2000,32:11-16
38. Osawa M, Hanada K, Hamada H, et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Science. 1996,273:242-245
39. Satdarshan P.S.Monga, Yi Tang, Fabio Candotti, et al. Expansion of hepatic and hematopoietic stem cell utilizing mouse embryonic liver explant. Cell Transplantation. 2001,10:81-89
40. Terai S, Sakaida I, Yamamoto N, et al. An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes. J Biochem (Tokyo). 2003,134(4):551-8.
41. Pittenger MF,et al. Multilineage potential of adult human mesenchymal stem cell. Science. 1999,284:143-147
42. Morayma Reyes, M,roy Lund,Catherine M.Verfaile et al. Purification and ex vivo expansion of postnatal human marrow mesodermal rogenitor cells. Blood 2001,98:2615-2625
43. Morayma Reyes,Arkadiusz Dudek, Catherine M. Verfaillie,et al. Origin of endothelial progenitors in human post-natal bone marrow. J.Clin Invest. 2002,109:337-346
44.Robert E.Schwartz,Morayma Reyes,Catherine M.Verfailie,et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest. 2002,109:1291-1302
45.Yuehua Jiang,Balkrishna N.Jahagirdar, Catherine M.Verfailie,et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002,418(6893):41-49
46.Yuehua Jiang, Ben Vaessen, Catherine M.Verfailie, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow,muscle,and brain. Exp Hematol. 2002, 30:896-904
47.Morayma Reyes, Catherine M.Verfailie. Characterization of Multipotent adult progenitor cells, a subpopulation of mesenchymal stem ceils. Ann. NY Acad. Sci. 2001,938:231-235
48.Wagers AJ, Sherwood RI, Christensen JL, et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002, 297(5590):2256-2259.
49. Friedenstein A J. Precursor cells of mechanocytes. Int Rev
Cytol. 1976,47:327-355
50. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subculativation and following cryopreservation Cell Biochem. 1997,64(2):278.
51. Pot Phillips. New surgical approaches to Parkonson disease. 美国医学杂质中文版. 2000,19(2):96-97
52. Wynter EA, Coutinho LH, Marsh JC,et al. Comparison of purity and enrichment of CD34+ cell of bone marrow, umbilical cord and peripheral blood (primed of apheresis) using five separation systems. Stem Cells. 1995,13:524-532
53. Pittenger M F,Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999,284 (5411): 143-147.
54. Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA. 2001,98(14): 7841-7845.
55．张春雨，段恩奎，曾国庆．白血病抑制因子及其在胚胎发育和胚泡着床中的生理功能．生理学进展．1997，28：240-242
56.Yamamoto N, Terai S, Ohata S,et al.A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver.Biochem Biophys Res Commun. 2004, 313(4):1110-1118.
57. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000,6:1229-1234
58. Oh SH, Miyazaki M, Kouchi H, et al. Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun. 2000,279(2):500-504
59. Okumoto K, Saito T, Hattori E,et al.Differentiation of bone marrow cells
into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation. Biochem Biophys Res Commun. 2003,304(4):691-5.
60. Michalopoulos GK, DeFrances MC. Liver regeneration. Science. 1997; 276 (5309):60-6
61.Weng YS, Lin HY, Hsiang YJ, et al.The effects of different growth factors on human bone marrow stromal cells differentiating into hepatocyte-like eells.Adv Exp Med Biol. 2003,534:119-28
62. Edith Arany, David J.Hill. Ontogeny of fibroblast growth factor in the early development of the rat endocrine pancreas. Pediatric Res. 2000, 48:389-403
63. Koh K, Ohta K, Nakamae H, et al. Differential effects of fibroblast growth factor-4, epidermal growth factor and transforming growth factor-betal on functional development of stromal layers in acute myeloid leukemia. Leuk Res. 2002,26:933
64. Leffert HL, Koch K S, Moran T. Hormonal control of rat liver regeneration. Gastroenterology. 1979,76:1470-1479
65．唐望先，张国，王俊平等．流式细胞术动态检测肝大部分切除后再生相关因子的实验研究．同济医科大学学报．2001，30：330-332
66．李玉昌，马军，林俊堂等．大鼠短间隔连续部分肝切除后ADAMs mRNA表达分析．胃肠病学和肝病学杂志．2001，10：235-237
67. Stone A L, Kroeger M, Sang Q X A, et al. Structure-function analysis of the ADAM family of disintegrin-like and metalloproteinase-containing protein. J protein chen. 1999, 18(4):447-465
68. Grompe M, Laconi E, Shatritz DA. Principles of therapeutic liver repopulation. Semin Liver Dis. 1999,19:7-14
69. Laconi S, Curreli F, Dianna S, et al. Liver regeneration in response to partial hepatectomy in rats treated with retrorsine:a kinetic study. J Hepatol. 1999, 31:1069-1074
70．陈柯，高毅，潘玉仙等．有效原代猪肝细胞培养体系的研制．世界华人消化杂志．1999，7(3)：206-209