供体骨髓输注诱导器官移植后特异性免疫低反应及机制
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摘要
器官移植是治疗终末期器官功能衰竭的一种有效的治疗方法,以CsA为代表的多种新型免疫抑制剂的出现使器官移植短期成功率得以提高,但伴随全面免疫抑制而发生的严重感染、肿瘤等并发症以及慢性移植物失功,严重影响了移植器官的长期生存率。如何提高移植器官的长期生存率是目前器官移植领域的一个研究热点,诱导对同种异基因移植物的免疫耐受可能是解决这一问题的较好方法,在啮齿类动物已有多种方法获得成功,其中,供体的骨髓细胞(Donor bone marrow cells, DBMC)在诱导免疫耐受中具有重要作用,但目前多种方案均有不同程度的缺点,大多与DBMC输注前需要进行严格的预处理(亚致死剂量照射、细胞毒药物等)作清髓或部分清髓,具有较大副作用有关,或者须在移植前输注DBMC,因此难于在以尸体供体为移植器官主要来源的临床工作中推广。近来有实验和临床研究显示在常规使用免疫抑制剂的状况下给予DBMC输注能减少急性排斥反应的发生和提高移植物的长期生存率,机理尚不明了。我们采用在短疗程CsA治疗下,术毕2次输注DBMC这一目前易被临床接受的方案,了解供体骨髓主要细胞成分能否延长同种异基因移植心脏的存活时间及其机理。
To date, the CsA-contained immunosuppressive regimen have effectively prevented acute rejection following organ transplantation, and improved the results of the short-term of the transplantation. However, there are plenty evidences that long-term CsA therapy results chronic CsA nephrotoxicity and chronic allograft nephropathy. Morbidity and mortality in graft recipients remain high. This led the chronic graft dysfunction at end and questioned its long- term therapy in organ transplantation. Thus, minimal calcineurin inhibitors such as CsA is the tendency of the development of an optimal immunosuppressant regimen.In an attempt to induce donor specific immunologic low-reaction in organ transplantation, a number of protocols have been attempted, and some yield ideal results in rodents. Among these protocols, the donor bone marrow cells (DBMC) infusion was emphasis. But DBMC infusion sometimes resulted severe toxic side effects and to date little is known about its mechanism.In this study, we investigate the synergistic action of donor bone marrow and short-term CsA on prolonging cardiac allograft survival in the rat cardiac transplantation model.
Part 1 Establishment and comparison of 2 types of rat heterotopic heart transplantationAim To study the advantage and disadvantage of 2 types of rat heterotopic heart transplantation model.Methods All experimental procedures were performed by one surgeon. The iso-strains transplantation were between BN rat to BN rat and the hetero-transplantation were between Lewis rat to BN rat. The aorta and pulmonary artery of donor's heart with the abdominal aorta and inferior vena cava of the receptor were anastomosed, respectively, in the heterotopic abdominal heart transplantation and with the right common carotid artery and the right external jugular vein of the receptor were anastomosed respectively in the heterotopic cervical heart transplantation. The function of the graft was followed up daily by palpation of heart beats. Pathological examination was performed at various time after surgery. Comparison was made on the operation time, operation success rate, and allografts survival time.Results The two types of operation (the heterotopic cervical heart transplantation and the heterotopic abdominal heart transplantation) were performed 100 times, respectively. Failure in the former group was mainly due to artery constriction and in the latter group was bleeding. Success rates of rat celiac and cervical heart transplantation in the formulate 40 times experiments were 85% (17/20) and 90% (18/20), respectively. The operation times in the two groups were not different (73.85±6.43min vs 70.7±8.67min. p=0.199). The survival times of the iso-allografts were longer than 100 days and not rejection. There are no significantly differences in the degree of acute rejection at various time after surgery on the pathological examination between the 2 types of rat heterotopic allo-heart transplantation.
Conclusions Establishment the 2 types of model were easy operation, short duration of the operation and high rate of success and may use in experimental study widely. The selection of rat heart transplantation depends upon the requirement of research. Part 2 Purify major cells from rat bone marrow cells and analyze their immunologic regulatory effectsAim To isolated donor rat bone marrow cells(DBMC), mesenchymal stem cells(MSC) and immature dendritic cells(imDC) from the rat bone marrow and analyzed their immunologic regulatory effects.Methods The rat DBMC, MSC and imDC from rat femora marrow cells were separated and purified by gradient centrifugation and adherence to the culture protocol. The morphological character of the cultured MSC and imDC was observed under light microscope and electron microscope. Expression of CD molecules were detected by flow cytometry. The isolated DBMC, MSC and imDCs were co-cultured with allogenic spleen cells in the mixed lymphocytes reaction, respectively. The spleen cells proliferation was measured by MTT colorimetry.Results The isolated DBMC, MSC and imDCs had the t
To date, the CsA-contained immunosuppressive regimen have effectively prevented acute rejection following organ transplantation, and improved the results of the short-term of the transplantation. However, there are plenty evidences that long-term CsA therapy results chronic CsA nephrotoxicity and chronic allograft nephropathy. Morbidity and mortality in graft recipients remain high. This led the chronic graft dysfunction at end and questioned its long- term therapy in organ transplantation. Thus, minimal calcineurin inhibitors such as CsA is the tendency of the development of an optimal immunosuppressant regimen.In an attempt to induce donor specific immunologic low-reaction in organ transplantation, a number of protocols have been attempted, and some yield ideal results in rodents. Among these protocols, the donor bone marrow cells (DBMC) infusion was emphasis. But DBMC infusion sometimes resulted severe toxic side effects and to date little is known about its mechanism.In this study, we investigate the synergistic action of donor bone marrow and short-term CsA on prolonging cardiac allograft survival in the rat cardiac transplantation model.
Part 1 Establishment and comparison of 2 types of rat heterotopic heart transplantationAim To study the advantage and disadvantage of 2 types of rat heterotopic heart transplantation model.Methods All experimental procedures were performed by one surgeon. The iso-strains transplantation were between BN rat to BN rat and the hetero-transplantation were between Lewis rat to BN rat. The aorta and pulmonary artery of donor's heart with the abdominal aorta and inferior vena cava of the receptor were anastomosed, respectively, in the heterotopic abdominal heart transplantation and with the right common carotid artery and the right external jugular vein of the receptor were anastomosed respectively in the heterotopic cervical heart transplantation. The function of the graft was followed up daily by palpation of heart beats. Pathological examination was performed at various time after surgery. Comparison was made on the operation time, operation success rate, and allografts survival time.Results The two types of operation (the heterotopic cervical heart transplantation and the heterotopic abdominal heart transplantation) were performed 100 times, respectively. Failure in the former group was mainly due to artery constriction and in the latter group was bleeding. Success rates of rat celiac and cervical heart transplantation in the formulate 40 times experiments were 85% (17/20) and 90% (18/20), respectively. The operation times in the two groups were not different (73.85±6.43min vs 70.7±8.67min. p=0.199). The survival times of the iso-allografts were longer than 100 days and not rejection. There are no significantly differences in the degree of acute rejection at various time after surgery on the pathological examination between the 2 types of rat heterotopic allo-heart transplantation.
Conclusions Establishment the 2 types of model were easy operation, short duration of the operation and high rate of success and may use in experimental study widely. The selection of rat heart transplantation depends upon the requirement of research. Part 2 Purify major cells from rat bone marrow cells and analyze their immunologic regulatory effectsAim To isolated donor rat bone marrow cells(DBMC), mesenchymal stem cells(MSC) and immature dendritic cells(imDC) from the rat bone marrow and analyzed their immunologic regulatory effects.Methods The rat DBMC, MSC and imDC from rat femora marrow cells were separated and purified by gradient centrifugation and adherence to the culture protocol. The morphological character of the cultured MSC and imDC was observed under light microscope and electron microscope. Expression of CD molecules were detected by flow cytometry. The isolated DBMC, MSC and imDCs were co-cultured with allogenic spleen cells in the mixed lymphocytes reaction, respectively. The spleen cells proliferation was measured by MTT colorimetry.Results The isolated DBMC, MSC and imDCs had the t
引文
1 The International Society for Heart Transplantation. A working formulation for the standardization of in the diagnosis of nomenclature heart and lung rejection: heart rejection study group. J Heart Transplant, 1990, 9: 587-593.
2 孙敬方 主编.动物实验方法学.人民卫生出版社.北京:2001年11月第1版.第6章 动物实验外科操作技术与常见手术方法 340
3 Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg. 1969, 57: 225-229.
4 邵孟平,吴清玉,王春生.改良大鼠同种异位心脏移植术.中国比较医学杂志,2003,13 (5):305-6.
5 李剑锋,彭志海,祝哲诚,蔡勇.改良的大鼠异位心脏移植术.上海实验动物科学,2003,23 (3):157-159.
6 Abbott CP. A technique for heart transplantation in the rat. Arch Surg. 1964. 89: 645.
2 孙敬方 主编.动物实验方法学.人民卫生出版社.北京:2001年11月第1版.第6章 动物实验外科操作技术与常见手术方法 340
3 Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg. 1969, 57: 225-229.
4 邵孟平,吴清玉,王春生.改良大鼠同种异位心脏移植术.中国比较医学杂志,2003,13 (5):305-6.
5 李剑锋,彭志海,祝哲诚,蔡勇.改良的大鼠异位心脏移植术.上海实验动物科学,2003,23 (3):157-159.
6 Abbott CP. A technique for heart transplantation in the rat. Arch Surg. 1964. 89: 645.