摘要
背景
肺移植是终末期肺病唯一有效的治疗方法。尽管目前肺移植的术后一年生存率可达71%,约46%的患者术后生存期可超过5年,但真正限制肺移植患者长期生存的原因仍然是移植术后的慢性排异,其最终结果即闭塞性细支气管炎(Obliterans Bronchiolitis, OB)的发生。国外的大宗资料统计表明,在生存期超过3月的病人中,有28%以上的患者存在着不同程度的OB;移植术后第一年OB发生率为50%。更长期的随访资料提示,在肺移植术后5到10年后,OB的发生率将高达60-80%。它不单可以直接导致死亡,同时也可以通过继发感染、以及相关治疗所引起的各类不良反应等间接导致患者死亡。匹兹堡医学中心的经验认为近25-40%的患者直接或者间接病死于OB。
目前预防和治疗移植后排斥反应的主要手段是应用免疫抑制药物。这些药物除需终生服用外,其毒副作用也非常明显,且价格昂贵,部分患者甚至因药物的毒副作用或经济负担被迫停药;同时此类免疫抑制治疗OB的方法往往疗效不理想,严重影响移植患者的长期存活。因此,研究免疫抑制或耐受的有效方法,对预防和治疗慢性排异的发生、进一步延长移植物存活具有至关重要的意义。
近年来随着研究的不断深入,临床免疫学家发现器官移植后通过向受体移植供体造血干细胞或骨髓来源的间充质干细胞(bone marrow mesenchymal stem cells, BM-MSCs)建立混合嵌合体,对免疫耐受的诱导有帮助。这一理论的提出解决了困绕移植学者多年的免疫排斥难题,成为目前移植领域研究的新前沿。
BM-MSCs是成体干细胞的一种,存在于骨髓中,具有干细胞的共性和很强的自我增殖能力及多向分化潜能,能表达多种表面抗原和分泌多种细胞因子,是一种较原始的骨髓基质细胞。研究发现经非清髓处理后,骨髓间充质干细胞( Bone marrow mesenchymal stem cells, BM-MSCs )输注受体后,髓系树突状细胞(DC)会定向迁移至胸腺并向T细胞提供阴性选择信号,有可能产生外周免疫耐受,这为抑制慢性排异提供了可能。
综上所述,我们构想将骨髓细胞移植用于诱导受体嵌合,并进一步诱导肺移植慢性排异的免疫抑制或免疫耐受,并对肺移植慢性排异的机理进行探讨。
方法
1、实验分两组:实验组(I组)采用10只SD大鼠为供体,10只Wistar大鼠为受体;对照组(II组)10只供体、受体均采用SD大鼠。均行异位气管移植。术后3,14,28天,处死大鼠,取出移植物。镜下观察:气道闭塞情况;气道上皮完整性;气道粘膜下炎细胞浸润、纤维组织增生的情况。
2、实验分7组( n=10 ):A组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次,7天后直接行大鼠气管异位移植,移植后4h内经尾静脉输注生理盐水0.8mL。B组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次。7天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。C组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次。7天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。D组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经尾静脉输注生理盐水0.8mL。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。E组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。F组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。G组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。移植后6天经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。随后测定异基因嵌合体量的变化及单向混合淋巴细胞培养的刺激效应,流式细胞仪检测了T淋巴细胞亚群的变化,检测细胞移植后受体内IL-2、IL-10细胞因子的表达和气管的阻塞率。检测移植物病理组织学,并进行统计学分析。
结果
1、两组移植气管及支气管的闭塞性细支气管炎发生率实验组闭塞性细支气管炎的发生率为100%,对照组未发生闭塞。对照组与实验组移植气管及支气管闭塞性细支气管炎发生率差异具有显著性意义
2、B、C组动物接受60Coγ射线全身照射经环磷酰胺预处理后行BMC/BM-MSCs移植, E、F组动物接受FK506腹腔注射后行BMC/BM-MSCs移植,在外周血、脾脏中均检测到嵌合的存在。输注BMC组较BM-MSCs组嵌合体水平维持更久。A、D组动物未接受BMC/BM-MSCs移植的未检测到嵌合。但嵌合体模型随着时间推移,其嵌合会渐渐消失。
3、于气管移植后21天后取雌性Wistar大鼠脾淋巴细胞作为效应细胞,用雄性SD脾淋巴细胞经丝裂霉素处理后作为刺激细胞将两者行单向混合细胞培养, A、B、C、D、E、F、G组与O组相比均呈增殖反应。计算刺激效应结果显示:C组较B组和A组的受体淋巴细胞增殖显著减弱(P<0.05);B组较A组也显著减弱(P<0.05)。G组较D、E、F组的受体淋巴细胞增殖显著减弱(P<0.05);F组较E组也显著减弱(P<0.05), E、F组较同期B、C组无明显差异。
4、大鼠接受5Gy全身照射后外周淋巴细胞于照后一周左右降至最低水平,经辐照后白细胞中T细胞亚群CD3+细胞比例急剧减低降至5%左右,但各处理组中CD3+细胞中CD4+、CD8+含量比值并无大的变化,只是与正常大鼠相比CD4+有所降低,而CD8+的含量稍有上升。
5、受体IL-2水平在无处理组(A、D组)气管移植后28天时较移植前明显升高(P<0.05),而在实验组(B、C、E、F、G组)移植术后IL-2水平较移植前也有升高,与A、D组比较有统计学差异(P<0.05)。无处理组受体IL-10水平在移植后28天时较移植前无明显差别(P>0.05),实验组(B、C、E、F、G组)在移植后28天时明显升高,与A组、D组比较有统计学差异(P<0.05)。E、F组较同期B、C组无明显差异。
6、BMC移植组和BM-MSCs移植组气管移植后28天阻塞率较对照组减低。移植后28天阻塞率E、F组较同期B、C组减低。
7、A、D组气管纤毛比例远低于正常气管和G组(P< 0.01),G组气管和正常气管相比无统计学意义。B组和E组相比无统计学意义, C组和F组相比无统计学意义。
8、于术后28天分批处死动物,在对照组标本中可以观察到严重的结缔组织包裹。在低倍镜下观察,对照组标本的气管管腔内有肉芽组织向管腔内增生,造成管腔闭塞。镜下观察可见对照组组在术后28天移植气管内有大量炎性细胞浸润,上皮层有坏死、脱落,可见有大量肉芽组织向管腔内生长,气管管腔几乎完全闭塞。而在使用BMC/BM-MSCs干预下,上述情况有所改善。见管腔通畅,上皮细胞少量坏死、脱落,覆盖于移植气管内壁,气管壁内可见少量炎细胞浸润。但B、C、E、F、G各组差别不明显。
结论
1.大鼠气管异位移植模拟肺移植慢性排斥反应。
2.新型的非破坏骨髓的预处理与传统的照射等非清髓性预处理方式比较同样诱导出了嵌合,并且在抑制慢性排斥反应方面同样有效。
3.通过在非清髓性预处理后输注供体骨髓细胞或骨髓间充质干细胞,可以建立混合嵌合体,调节Th1/Th2型细胞因子的表达,使其向免疫耐受方向偏移,可以形成对肺移植慢性排斥反应的供体特异性的免疫抑制。
Background:
Lung transplantation is the most important method in healing the serious pulmonary disease. The advances in immunosuppressive drug have yielded improvement of transplantation; yet long-term survival recipient complications and graft loss continue to accompany chronic immunosuppression. Induction of tolerance may hold the key to improve transplant longevity and quality of life in recipient and increase the cost-effectiveness of transplant therapy at the same time. With further studies and developments of transplantation immunology, eliminating transplant rejection through inducing immune tolerance has become the main goal in organ transplantation. Obliterative bronchiolitis(OB) is the most important cause of graft dysfunction following lung transplantation.
The purpose of this study was to induce chimera by donor bone marrrow and mesenchymal stem cells transplantation and observe its effects on obliterative bronchiolitis(OB) after lung transplantation, and investigate the pathogenesis of transplantation tolerance of chimera preliminarily in an animal model.
Methods:
1、SD and Wistar rat were, respectively, selected as donor and recipient In experimental group, while in control group, SD rat served as donors and recipients, respectively.All grafts were harvested 28 days after transplantation and stained with hematoxylin and eosin (HE) to observe the histopathology of the grafts. OB incidence in two groups was compared.
2、SD and Wistar rat were, respectively, selected as donor and recipient.Donor and recipient were divided randomly into 8 groups. Recipient was conditioned with sublethal whole body irradiation (WBI) in Group A, B and C. Group A infused with saline solution; Group B infused with bone marrow cells (BMC) of SD rat; Group C infused with bone marrow mesenchymal stem cells (BM-MSCs) of SD rat. Animals in other groups did not receive whole body irradiation, they received FK506 3 mg two days before transplantation. Then Group D infused with saline solution. Group E infused with bone marrow cells (BMC) of SD rat. Group F infused with bone marrow mesenchymal stem cells (BM-MSCs) of SD rat. Group G infused with bone marrow mesenchymal stem cells (BM-MSCs) and bone marrow cells (BMC) of SD rat. The tracheal segments of Wistar rat were then heterotopically transplanted into recipient rats in all groups. Grafts were harvested at day 3, 14 ,28 after transplantation. To explore immune depression mechanisms, the mixed lymphocyte reaction (MLR) was performed. The level of IL-2、IL-10 and the obliteration ratio were also measured. Recipient rats were detected for donor origin cells in the peripheral blood lymphocyte and spleen by polymerase chain reaction (PCR). All grafts were harvested 28 days after transplantation and stained with hematoxylin and eosin (HE) to observe the histopathology of the grafts.
Results:
1、The experimental group showed the histopathology of OB. The incidence of OB in the experimental group was significantly lower than in the control group.
2、The results showed that donor lymphoid chimeras can be found in the immune depression Wistar rat (Group C > Group B). Wistar rat were specifically tolerant to the SD rat in MLR assay in Group B and Group C. The obliteration ratio of allograft in group A was higher than that in group B and C( P<0.05).
3. Chimera can be found in the peripheral blood and spleen lymphocyte in the tolerance Wistar rats of Group B, C, E, F, and G.
4. Allografts in Groups infused with bone marrow mesenchymal stem cells (BM-MSCs) / bone marrow cells (BMC) of SD rat expressed significantly less IL-2 expression than that in Group A. 5.At the same time, lymphocytic infiltration and luminal obliteration were lower in Groups infused with bone marrow mesenchymal stem cells (BM-MSCs) / bone marrow cells (BMC) of SD rat than that in Group A and D (p<0.01).
Conclusions:
1、Heterotopic tracheal allografting in rat as a model of chronic allograft rejection after lung transplantation displays many advantages.
2、These results suggest that non-myeloablative conditioning regimens for allogeneic BMC/BM-MSCs transplantation can successfully establish mixed chimeras model from SD to Wistar rat and induce a specific immune depression in Wistar rat .
引文
1. Arcasoy SM, Kotloff RM. Lung transplantation. N Eng J Med. 1999; 340:1081–1091
2. Al-Githmi I, Batawil N, Shigemura N. Bronchiolitis obliterans following lung transplantation. Eur J Cardiothorac Surg. 2006; 30: 846-51.
3. Nicod LP. Mechanisms of airway obliteration after lung transplantation. Proc Am Thorac Soc. 2006; 3(5): 444-9.
4. Olivier B, Gabriel T, Yves C, et al. Lung Retransplantation for bronchiolitis obliterans syndrome: long-term follow-up in a series of 15 recipients. Chest. 2003: 1832-1837
5. Weiss MJ, Madsen JC, Rosengard BR, et al. Mechanisms of chronic rejection in cardiothoracic transplantation. Front Biosci. 2008; 1(13): 2980-8.
6. Neuringer, Isabel P.; Aris, Robert M.; Burns, Kim A, et al. Epithelial Kinetics in Mouse Heterotopic Tracheal Allografts.American Journal of Transplantation. 2002, 2(5): 410-419.
7. Piccotti JR, Li K, Chan SY, Eichwald EJ, Bishop DK. Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process. Transplantation. 1999, 67(12): 1548-1555.
8. K?hler CM, Wechselberger J, Hilbe W, et al. Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury. Respir Res. 2007; 9(8): 50.
9. Kawahara T, Shimizu I, Ohdan H, et al. Differing mechanisms of early and late B cell hyporesponsiveness induced by mixed chimerism. Am J Transplant. 2005; 5(12): 2821-9.
10. Billiau, An D, Sefrioui, Hassane, et al. Transforming growth factor-βinhibits lymphokine activated killer cytotoxicity of bone marrow cells: implications for the graft-versus-leukemia effect in irradiation allogeneic BOne marrow chimeras. Transplantation. 2001; 71(2): 292-9.
1. Mal H, Sleiman C , Roue C, et al .Selection criteria of candidates for lung transplantation .Rev Mal Respir,1997,14:423-429
2. Hosenpud JD, Bennet LE, Keck BM, et al.The registry of the international society for heart and lung transplantation:Fifteenth Official Report-1998. J Heart Lung Transplant1998,17:656–668.
3. Dosanjh A, Wan B, Ikonen T, et al. Airway goblet cells and respiratory epithelial injury in an animal model of obliterative airways disease (OAD). Am J Transplant 2001, 1: 321–324.
4. Jaramillo A, Smith CR, Maruyama T, et al. Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome. Hum Immunol 2003, 64: 521–529.
5. Adams BF, Brazelton T, Berry GJ, et al. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation, 2000, 69(4):661-665.
6. Asimacopoulos PJ.Molokhia FAS.Pegg CAS Lung transplan-rationinthe rat 1971(1)
7. Qu N, d Vos P, Schelfhorst M, et al. Integrity of airway epithelium is essential against obliterative airway disease in transplanted rat tracheas. J Heart Lung Transplantation. 2005,24(7):882-890.
1. Arcasoy SM, Kotloff RM. Lung transplantation. N Eng J Med. 1999; 340:1081–1091
2. Al-Githmi I, Batawil N, Shigemura N. Bronchiolitis obliterans following lung transplantation. Eur J Cardiothorac Surg. 2006; 30: 846-51.
3. Nicod LP. Mechanisms of airway obliteration after lung transplantation. Proc Am Thorac Soc. 2006; 3(5): 444-9.
4. Olivier B, Gabriel T, Yves C, et al. Lung Retransplantation for bronchiolitis obliterans syndrome: long-term follow-up in a series of 15 recipients. Chest. 2003: 1832-1837
5. Weiss MJ, Madsen JC, Rosengard BR, et al. Mechanisms of chronic rejection in cardiothoracic transplantation. Front Biosci. 2008; 1(13): 2980-8.
6. Neuringer, Isabel P.; Aris, Robert M.; Burns, Kim A, et al. Epithelial Kinetics in Mouse Heterotopic Tracheal Allografts.American Journal of Transplantation. 2002, 2(5): 410-419.
7. Piccotti JR, Li K, Chan SY, Eichwald EJ, Bishop DK. Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process. Transplantation. 1999, 67(12): 1548-1555.
8. K?hler CM, Wechselberger J, Hilbe W, et al. Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury. Respir Res. 2007; 9(8): 50.
9. Kawahara T, Shimizu I, Ohdan H, et al. Differing mechanisms of early and late B cell hyporesponsiveness induced by mixed chimerism. Am J Transplant. 2005; 5(12): 2821-9.
10. Billiau, An D, Sefrioui, Hassane, et al. transforming growth factor-βinhibits lymphokine activated killer cytotoxicity of bone marrow cells: implications for the graft-versus-leukemia effect in irradiation allogeneic BOne marrow chimeras. Transplantation. 2001; 71(2): 292-9.
11. Hale, Douglas A.; Gottschalk, Rita, et al. Immunologic mechanisms in tolerance produced in mice with nonradiation-based lymphoablation and donor-specific bone marrow. [J] Transplantation. Aug 27, 2002, 74(4):477-484.
12. Neipp M , Exner BG , Ildstad ST. et al. A nonlethal conditioning approach to achieve engraftment of xenogeneic rat marrow in mice and to induce donor-specific tolerance. [J] Transplantation, Oct 27, 1998 , 66(8) :969– 975.
13. Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. Journal of Embryology & Experimental Morphology, 1966, 16 (3):381-390
14. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Experimental Biology & Medicine, 2001, 226(6): 507-520
15. Deans RJ, Moseley AB, Mesenchymal stem cells: biology and potential clinical uses. Experimental Hematology, 2000 ,28(8):875-884
16.侯树勋,孙大铭,杜桂鑫,等.骨形成蛋白-7真核表达质粒的构建及在骨髓间充质干细胞中的表达.中华外科杂志, 2003, 41: 445-448.
17. Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997, 90(9):3471-3481
18. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP. Gowen M. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications forosteopenic disorders. Journal of Bone & Mineral Research, 1998, 13(3):371-382
19. Ghilzon R, McCulloch CA, Zohar R. Stromal mesenchymal progenitor cells. Leukemia & Lymphoma, 1999, 32(3-4):211-221
20. Justesen J, Stenderup K, Eriksen EF, Kassem M. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures. Calcified Tissue International, 2002, 71(1):36-44
21. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147
22. Majumdar MK, Thiede MA ,Mosca JD, Moorman M, Gerson SL. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. Journal of Cellular Physiology, 1998, 176(1):57-66
23. Haga, M; Tsuchida, M;Hirahara, et al. Synergistic effect of anti-T cell receptor monoclonal antibody and 15-deoxyspergualin on cardiac xenograft survivalin a mouse-to-rat model. [J]Transplantation, 2000, 69(12):2613-2621
24. Pawson, R.; Potter, M. N, et al Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant. [J] British Journal of Haematology. 2001, 115(3):622-629.
25. Fuchimoto Y. Huang CA. et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. [J] Journal of Clinical Investigation. Jun, 2000. 105(12):1779-89.
26. Wang, Jia-Wang; Howson, Julie M., et al. Influence of SHIP on the NK Repertoire and Allogeneic Bone Marrow Transplantation. [J] Science. Issues in Public Health. 2002, 295(5562):2094-2097.
27. Chen Ann M, Zhou Y, Swenson K. et al. Porcine stem cell engraftment and seeding of murine thymus with class II cells in mice expression porcine cytokines toward tolerance induction across discordant xenogeneic barriers.[J] Transplantation, 2000, 69(12):2 484- 2 490.
28. Jiang, Yuehua; Jahagirdar, Balkrishna N. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. [J] Nature. 2002, 418(6893):41-49.
29. Devine, Steven M.; Link, Dan; et al. Novel strategies for hematopoietic stem cellmobilization. [J] Current Opinion in Organ Transplantation. 2004, 9(1):54-63.
30. Li, Hua; Inverardi, Luca; Molano, R, et al. Camillo Nonlethal conditioning for the induction of allogeneic chimerism and tolerance to islet allografts. [J] Transplantation. 2003, 75 (7):966-970.
31. Storb R, Champlin R, et al. Nor-myeloabalative transplants for malignant disease. In: Hematology 2001 The American Society of Hematology Education Program Book, 2001: 375-391.
32. Neipp M , Exner BG , Ildstad ST. et al. A nonlethal conditioning approach to achieve engraftment of xenogeneic rat marrow in mice and to induce donor-specific tolerance. [J] Transplantation, 1998 , 66(8) :969– 975.
33.夏穗生.临床移植医学.第一版,浙江科学技术出版社.1999:9
34.陈实.移植免疫学.第一版,湖北科学技术出版社.1998:256
35. Devine, Steven M.; Link, Dan; et al. Novel strategies for hematopoietic stem cell mobilization. [J] Current Opinion in Organ Transplantation. 2004, 9(1):54-63.
1. Arcasoy SM, Kotloff RM. Lung transplantation. N Eng J Med. 1999; 340:1081–1091
2. Al-Githmi I, Batawil N, Shigemura N. Bronchiolitis obliterans following lung transplantation. Eur J Cardiothorac Surg. 2006; 30: 846-51.
3. Nicod LP. Mechanisms of airway obliteration after lung transplantation. Proc Am Thorac Soc. 2006; 3(5): 444-9.
4. Olivier B, Gabriel T, Yves C, et al. Lung Retransplantation for bronchiolitis obliterans syndrome: long-term follow-up in a series of 15 recipients. Chest. 2003: 1832-1837
5. Weiss MJ, Madsen JC, Rosengard BR, et al. Mechanisms of chronic rejection in cardiothoracic transplantation. Front Biosci. 2008; 1(13): 2980-8.
6. Genden EM,Iskander AJ,Bromberg JS.Orthotopic tracheal allografts undergo reepithelialization with recipient-derived epithelium.Arch Otolaryngol Head Neck Surg 2003,129:118-123
7. Genden EM, Iskander A, Bromberg JS,et al. The kinetics and pattern of tracheal allograft re-epithelialization. Am J Respir Cell Mol Biol. 2003,28(6): 673–681.
8. Qu N, d Vos P, Schelfhorst M, et al. Integrity of airway epithelium is essential against obliterative airway disease in transplanted rat tracheas. J Heart Lung Transplantation. 2005,24(7):882-890.
9. Shaari CM,Farber D,Brandwein MS,et al. characterizing the antigenic profile of the human trachea:implications for tracheal transplantation[J]. Head Neck, 1998, 20: 522–27
10. Rosso F, Giordano A, Barbarisi M, et al. From cell-ECM interactions to tissue engineering. J Cell Physiol, 2004, 199: 174–80
11. Bach FH. Ferran C. et al. Accommodation of xenografts: expression of "protective genes" in endothelial and smooth muscle cells. [J] Transplantation Proceedings. Feb-Mar,1997. 29(1-2):56-8.
12. Thai NL,et al.Cytokine mRNA profiles in mouse orthotopic liver transplantation:graft rejection is assosiated with augmented TH1 fuction. Transplantation 1995;59:274-281
13. King MB;Jessurun J;Savik SK ,et al. Cyclosporine reduces development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation. 1997;63(4):274-281
1. Askari AT,Unzek S,Popovic ZB,et al.Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet, 2003,362:697-703.
2. Sttauet BE,Brehm M,Zeus T,et al.Intracoronary,human autologous stem cell transplantation for myocardial regeneration following myocardial infarction.Dtsch Med Wochenschr,2001,126:932-938.
3. Engelhardt JF,Yankaskas JR,Emst SA,et al.Submucosal glands are the predominant site of CFTR expression in the human bronchus.Nat Genet,1992,2:240-248.
4. Johnson LG,Olsen JC,Sarkadi B,et al.Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat Genet,1992,2:21-25.
5. Neuringer IP,Randell SH.Stem cell and repair of lung injuries.Respir Res,2004,5:6.
6. Ferrari G,Cusella-De Angelis G,Coletta M,et al.Muscle regeneration by bone marrow-derived myogenic progenitors.Science,1998,279:1528-1530.
7. Gussoni E,Soneoka Y,Strickland CD,et al.Dystrophin expression in the mdx mouse restored by stem cell transplantation.Nature,1999,401:390-394.
8. Orlic D,Kajstura J,Chimenti S,et al.Done marrow cells regenerate infarcted myocardium.Nature,2001,410:701-705.
9. Jackson KA,Majka SM,Wang H,et al.Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.J Clin Invest,2001,107:1395-1402.
10. Lin Y,Weisdorf DJ,Solovey A,et al.Origins of circulating endothelial cells and endotuelial outgrowth from blood.J Clin Invest,2000,105:71-77.
11. 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.
12. 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.
13. Krause DS,Theise ND,Collector MI,et al.Multi-organ,multi-lineage engraftment by a single bone marrow-derived stem cell.Cell,2001,105:369-377.
14. Brazelton TR,Rossi FM,Keshet GI,et al.From marrow to brain:expression of neuronal phenotypes in adult mice.Science,2000,290:1775-1779.
15. Mezey E,Chandross KJ,Harta G,et al.Turning blood into brain:cells bearing neuronal antigens generated in vivo from bone marrow.Science,2000,290:1779-1782.
16. Sanchez-Ramos J,Song S,Cardozo-Pelaez F,et al.Adult bone marrow stromal cells differentiate into neural cells in vitro.Exp Neurol,2000,164:247-256.
17. Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284:143-147.
18. Reyes M,Lund T,Lenvik T,et al.Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells.Blood,2001,98:2615-2625.
19. Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002,109:337-346.
20. Schwartz RE,Reyes M,Koodie L,et al.Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.J Clin Invest, 2002, 109: 1291-1302.
21. Jiang Y,Jahagirdar BN,Reinhardt RL,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2002,418:41-49.
22. Orkin SH,Morrison SJ.Stem-cell competition.Nature,2002,418:25-27.
23. Spencer H,Jaffe A.The potential for stem cell therapy in cystic fibrosis.J R Soc Med,2004,97:52-56.
24. Engelhardt JF,Schlossberg H,Yankaskas JR,et al.Progenitor cells of the adult human airway involved in submucosal gland development.Development,1995,121:2031-2046.
25. Zepeda ML,Chinoy MR,Wilson JM.Characterization of stem cells in human airway capable of reconstituting a fully differentiated bronchial epithelium.Somat Cell Mol Genet,1995,21:61-73.
26. Boers JE,Ambergen AW,Thunnissen FB.Number and proliferation of Clara cells in normal human airway epithdlium.Am J Respir Crit Care Med,1999,159:1585-1591.
27. Boers JE,Ambergen AW,Thunnissen FB.Number and proliferation of basal and parabasal cells in normal human airway epithdlium.Am J Respir Crit Care Med,1998,157:2000-2006.
28. Adamson IY,Bowden DH.Derivation of typeⅠepithelium from typeⅡcells in the developing rat lung.Lab Invest,1975,32:736-745.
29. Borthwick DW,Shahbazian M,Krantz QT,et al.Evidence for stem-cell niches in the tracheal epithelium.Am J Respir Cell Mol Biol,2001,24:662-670.
30. Kotton DN,Ma BY,Cardoso WV,et al.Bone marrow-derived cells as progenitors of lung alveolar epithelium.Development,2001,128:5181-5188.
31. Delplanque A,Coraux C,Tirouvanziam R,et al.Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice.J Cell Sci, 2000, 113:767-778.
32. Grove JE,Lutzko C,Priller J,et al.Marrow-derived cells as vehicles for delivery of gene therapy to pulmonary epithelium.Am J Respir Cell Mol Biol,2002,27:645-651.
33. Abe S,Lauby G,Boyer C,et al.Transplanted BM and BM side population cells contribute progeny to the lung and liver in irradiated mice. Cytotherapy, 2003, 5:523-533.
34. Ortiz LA,Gambelli F,McBride C,et al.Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.Proc Natl Acad Sci USA,2003,100:8407-8411.
35. Suratt BT,Cool CD,Serls AE,et al.Human pulmonary chimerism after hematopoietic stem cell transplantation.Am J Respir Crit Care Med,2003,168:318-322.
36. Kleeberger W,Versmold A,Rothamel T,et al.Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury.Am J Pathol,2003,162:1487-1494.
37. Terada N,Hamazaki T,Oka M,et al.Bone marrow cells adopt the phenotyhe of other cells by spontaneous cell fusion.Nature,2002,41:542-545.
38. Ying QL,Nichols J,Evans EP,et al.Changing potency by spontaneous fusion. Nature, 2002,416:545-548.
39. Wurmser AE,Gage FH.Stem cells:cell fusion causes confusion. Nature, 2002, 416: 485-487.
40. Charlton B,Auchincloss H Jr,Fathman CG.Mechanisms of transplantation tolerance. Annu Rev Immunol,1994,12:707
41. Bartholomew A,Sturgeon C,Siatskas M,et al.Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.Exp Hematol,2002,30:42
42. Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 2002,99:3838
43. Hashimoto F,Sugiura K,Inoue K,et al.Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vivo.Blood,1997,89:49
44. Beyth S,Borovsky Z,Mevorach D,et al.Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood, 2005,105:2214
45. Kawahara, T.; Shimizu, I.; Ohdan, H.; Zhao, G.; Sykes, M. et al. Differing Mechanisms of Early and Late B Cell Hyporesponsiveness Induced by Mixed Chimerism. [J] American Journal of Transplantation. , Dec, 2005. 5(12):2821-2829.
46. Billiau, An D.; Sefrioui, Hassane; et al. Transforming growth factor-βinhibits lymphokine activated killer cytotoxicity of bone marrow cells : Implications for the Graft-versus-Leukemia Effect in Irradiation Allogeneic Bone Marrow Chimeras. [J] Transplantation. Jan 27, 2001. 71(2):292-299.
47. Rachamim N, Gan J, Segall H, Krauthgamer R, Marcus H, Berrebi A. Martelli M, Reisner Y, Tolerance induction by "megadose" hematopoietic transplants: donor-type human CD34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture. Transplantation, 1998,65(10):1386-1393
48. Fuchimoto Y, Huang CA, Yamada K, Shimizu A, Kitamura H, Colvin RB, Ferrara V, Murphy MC, Sykes M, White-Scharf M, Neville DM Jr, Sachs DH. Mixed chimerism and tolerance without whole body irradiation in a large animal model.[see comment]. Journal of Clinical Investigation, 2000, 105(12):1779-1789
49. Fuchimoto Y. Huang CA. et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. [J] Journal of Clinical Investigation. Jun, 2000. 105(12):1779-89.
50.唐君玲,李晓明。骨髓间充质干细胞与骨髓移植后移植物抗宿主病的研究现状。泸州医学院学报。2006;29(2)163-165。
51.贺文凤,石庆之。间充质干细胞在造血干细胞移植中的免疫调节作用。江西医药。2006; 41(1)50-52。
52.魏宁,陈昶,高文,韩彪。干细胞诱导分化肺组织的研究进展。中华结核和呼吸杂志。2006;29(4)270-272。
1. Starzl TE. The "privileged" liver and hepatic tolerogenicity. [J] Liver Transplantation. Oct, 2001. 7(10):918-920.
2. Starzl TE. Murase N. et al. Microchimerism, macrochimerism, and tolerance. [J] Clinical Transplantation. Aug, 2000. 14(4 Pt 1):351-354.
3. Starzl TE. Acquired tolerance, allograft "acceptance," and immune suppression. [J] Transplantation Proceedings. May, 2000. 32(3):515.
4. Starzl TE. Chimerism and tolerance in transplantation. [J] Proceedings of the National Academy of Sciences of the United States of America. Oct 51, 2004. 01 Suppl 2:14607-14.
5. Starzl TE. Murase N. et al. Lessons of organ-induced tolerance learned from historical clinical experience. [J] Transplantation. Mar 27, 2004. 77(6):926-9.
6. Manilay, Jennifer O.; Pearson, Denise A.; Sergio, Justin J, et al. Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen. [J] Transplantation. Jul 15, 1998, 66(1):96-102.
7. Wekerle, Thomas; Sayegh, Mohamed H., et al Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematpoietic chimerism and tollerance.[J]Transplantation. Nov 15, 1999, 68(9):1348-1355.host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture. [J] Transplantation, 1998; 65: 1386-1393
19. Farg S S, Fehniger T A, Ruggeri L, et al. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. [J] Blood, 2002, 100:1935-1947
20. Reisner, Yair; Gur, Hilit; et al. Hematopoietic Stem Cell Transplantation across Major Genetic Barriers: Tolerance Induction by Megadose CD34 Cells and Other Veto Cells. [J] Annals of the New York Academy of Sciences. 2003, 996:72-79.
21. Millan, Maria T.; Shizuru, Judith A.; et al. Mixed chimerism and immunosuppressive drug withdrawal after hla-mismatched kidney and hematopoietic progenitor transplantation. [J] Transplantation. May 15, 2002. 73(9):1386-1391.
22. Ruggeri L, Capanni M, Urbaui E, et al. Effcctivcness of donor natural killer cell alloreactivitv in mismatched hematopoietic transplants. [J] Science, 2002. 295: 2097-2100
23. Fuchimoto Y. Huang CA. et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. [J] Journal of Clinical Investigation. Jun, 2000. 105(12):1779-89.
24. Kawai, Tatsuo; Benedict Cosimi, A.; et al. Effect of mixed hematopoietic chimerism on cardiac allograft survival in cynomolgus monkeys1. [J] Transplantation. Jun 15, 2002. 73(11):1757-1764.
25. Adams AB. Durham MM. et al. Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression. [J] Journal of Immunology. Jul 15, 2001. 167(2):1103-11.
26. Kean LS. Durham MM. et al. A cure for murine sickle cell disease through stable mixed chimerism and tolerance induction after nonmyeloablative conditioning and major histocompatibility complex-mismatched bone marrow transplantation. [J] Blood, Mar 1, 2002. 99(5):1840-9.
27. Foster RD. Fan L. Neipp M. et al. Donor-specific tolerance induction in composite tissue allografts. [J] American Journal of Surgery. Nov, 1998. 176(5):418-21.
28. Haynes BF. Markert ML. et al. The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection. [J] Annual Review of Immunology. 2000. 18:529-60.
29. Huang, Christene A.; Fuchimoto, Yasushi; et al. Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model. [J] Clinical Investigation. Jan 2000, 105(2):173-181.
30. Deans RJ. Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. [J] Experimental Hematology. Aug, 2000. 28(8):875-84.
31. Tse WT. Pendleton JD. Beyer WM. Egalka MC. Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. [J] Transplantation. Feb 15, 2003. 75(3):389-397.
32. Di Nicola M. Carlo-Stella C. et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. [J] Blood. May 15, 2002. 99(10):3838-3843.
33. Rasmusson I. Ringden O. et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. [J] Transplantation. Oct 27, 2003. 76(8):1208-1213.
34. Devine SM. Cobbs C. et al. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. [J] Blood. Apr 15, 2003. 101(8):2999-3001.
35. Sellers MT. Deierhoi MH. et al. Tolerance in renal transplantation after allogeneic bone marrow transplantation-6-year follow-up. [J] Transplantation. Jun 15, 2001. 71(11):1681-3.
36. Humblet-Baron S. Baron F.et al. Donor lymphocyte infusion for late relapse followed by kidney transplantation for end-stage renal failure after allogeneic bone marrow transplantation for chronic myeloid leukemia. [J] Transplantation. Nov 27, 2003. 76(10):1531-2.