骨髓移植诱导临床心脏移植后供者特异性免疫耐受方案的探讨

目的 探讨骨髓移植诱导临床心脏移植后供者特异性免疫耐受的可行性.方法 采取供心的同时采用改良"灌流法"获取供者的骨髓350 ml,经过滤及离心处理后,加入细胞冷冻保护液共80ml,分装于低温冻存袋,经程序降温,置于-80℃冰箱中保存.在常规原位心脏移植术后40 d,取冻存骨髓快速复温,穿刺受者双侧髂后上嵴,立即行骨髓腔内骨髓细胞输注(IBM-BMT),共输注单核细胞1.2×107/kg,CD34+细胞2.38×105/kg.骨髓输注前3 d行预处理,包括应用氟达拉滨、抗胸腺细胞球蛋白及全身淋巴结照射.骨髓移植后静脉应用他克莫司(Tac),维持血Tac浓度谷值在10～20μg/L;3周后改为口服Tac+吗替麦考酚酯(MMF);6周后改为环孢素A及MMF.分别于心脏移植后2、4、8和12周采集受者外周血,分别于术后4、8和12周采集受者的骨髓,应用短串联重复序列-聚合酶链反应法检测供者嵌合体.心脏移植后每周行心肌内心电图检查,每月行心肌活检1次.术后3个月,取受者及第三者外周血单核细胞,行混合淋巴细胞反应(MLR).结果心脏移植后1、2及3个月时受者的外周血及髂骨内骨髓细胞中供者来源的细胞比例分别为26.3%、19.1%、4.8%和46.3%、24.4%、7.6%.IBM-BMT后心肌内心电图监测显示心肌阻抗及R波波幅无明显变化.术后3个月行心内膜心肌活检,未见排斥反应征象.术后3个月时行超声心动图检查,提示心脏舒张、收缩功能良好.MLR提示受者对供者特异性刺激呈现低反应性,而对第三者仍保持良好的免疫活性(P＜0.01).结论 采取分期骨髓移植免疫耐受诱导方案可安全、有效地建立嵌合体,成功诱导心脏移植后供者特异性免疫耐受,但远期效果有待进一步研究.

Abstract：

Objective To investigate a new strategy of bone marrow transplantation (BMT) for donor-specific tolerance induction after heart transplantation. Methods Donor bone marrow cells (BMCs)were harvested simultaneously with donor cardiac graft using modified perfusion method (PM) ,then stored in a -80 ℃ refrigerator after filtration and centrifugation. Whole BMCs (IBM-BMT) (monocytes 1.2 ×107/kg,CD34+ cells 2.38× 105/kg) in host iliac bones were injected into the bone marrow cavity 40 days after heart transplantation. Preconditoning regimens that consisted of fludarabine, antithymoctye globin and total lymphoid irradiation were performed 3 days before BMT. Tacrolimus (Tac) was administrated intravenously after BMT or orally in conjunction with mycophenolate mofetil (MMF) 3 weeks later.Cyclosporine and MMF were orally administrated 6 weeks later. Donor chimerism was detected using short tandem repeats-polymerase chain reaction in monocytes from peripheral blood at the 2nd,4th, 8th or 12th week after BMT or BMCs at the 4th, 8th or 12th week after BMT. Intramyocardium electrocardiography examination or endomyocardial biopsy was performed weekly or monthly respectively. Mixed lymphocyte reactions (MLR) were performed 3 months after BMT. Results Donor chimerism in monocytes in peripheral blood or BMCs in iliac bones measured at the 1 st,2nd and 3rd month after BMT was 26.3%, 19.1%,4.8% ,and 46.3%, 24.4%, 7.6%, respectively. After 3-month follow-up, there was no rejection confirmed by endomyocardial biopsy or intramyocardium electrocardiography. Echocardiography revealed that the diastolic and systolic function of the cardiac graft was maintained well 3 months after BMT. MLR revealed donor-specific hyporesponsiveness while immunocompetence was preserved to third-party antigens. Conclusion These findings indicate that the two-stage BMT strategy is a safe and feasible method for the induction of donor-specific tolerance via stable mixed chimerism and needs to be further confirmed after a long-term observation.

A novel bone marrow transplantation strategy for donor-specific tolerance induction after heart transplantation

Objective To investigate a new strategy of bone marrow transplantation (BMT) for donor-specific tolerance induction after heart transplantation. Methods Donor bone marrow cells (BMCs)were harvested simultaneously with donor cardiac graft using modified perfusion method (PM) ,then stored in a -80 ℃ refrigerator after filtration and centrifugation. Whole BMCs (IBM-BMT) (monocytes 1.2 ×107/kg,CD34+ cells 2.38× 105/kg) in host iliac bones were injected into the bone marrow cavity 40 days after heart transplantation. Preconditoning regimens that consisted of fludarabine, antithymoctye globin and total lymphoid irradiation were performed 3 days before BMT. Tacrolimus (Tac) was administrated intravenously after BMT or orally in conjunction with mycophenolate mofetil (MMF) 3 weeks later.Cyclosporine and MMF were orally administrated 6 weeks later. Donor chimerism was detected using short tandem repeats-polymerase chain reaction in monocytes from peripheral blood at the 2nd,4th, 8th or 12th week after BMT or BMCs at the 4th, 8th or 12th week after BMT. Intramyocardium electrocardiography examination or endomyocardial biopsy was performed weekly or monthly respectively. Mixed lymphocyte reactions (MLR) were performed 3 months after BMT. Results Donor chimerism in monocytes in peripheral blood or BMCs in iliac bones measured at the 1 st,2nd and 3rd month after BMT was 26.3%, 19.1%,4.8% ,and 46.3%, 24.4%, 7.6%, respectively. After 3-month follow-up, there was no rejection confirmed by endomyocardial biopsy or intramyocardium electrocardiography. Echocardiography revealed that the diastolic and systolic function of the cardiac graft was maintained well 3 months after BMT. MLR revealed donor-specific hyporesponsiveness while immunocompetence was preserved to third-party antigens. Conclusion These findings indicate that the two-stage BMT strategy is a safe and feasible method for the induction of donor-specific tolerance via stable mixed chimerism and needs to be further confirmed after a long-term observation.