非清髓性预处理诱导小鼠心脏移植免疫耐受的研究

发布时间：2018-08-21 20:34

【摘要】： 目的:(1)通过比较各种不同的预处理方案,筛选出一种低毒性、适合临床实验、能诱导出长期稳定的供者特异性免疫耐受等优点的预处理方案,为进一步临床实验提供可靠的实验依据。(2)探讨移植早期残余宿主抗供者抗原的成熟T细胞在急性排斥反应、造血干细胞增殖与免疫耐受中的作用,以更好地指导建立有效、可行的诱导免疫耐受的方案。方法:(1)建立小鼠耳后心脏移植模型。全身X线照射(TBI)、注射抗胸腺细胞血清(ATS)与输注供者C57BL/6(H-2b)骨髓细胞(5.0×10~6)等不同组合,给予受者BALB/C(H-2d)小鼠进行不同预处理,共11组,然后进行心脏移植,术后不给任何免疫抑制治疗。10倍放大镜下观察心脏跳动情况,心脏跳动完全停止视为排斥反应。骨髓移植后28天常规检测外周血中嵌合体。(2)受体BALB/C小鼠分三组,分别给予TBI(800cGy)+ BMT、TBI(450cGy)+BMT、ATS+TBI(450cGy)+BMT三种不同的方案,分别于BMT后第7、第14、第21、第28、第42天检测其外周血、脾脏、胸腺中嵌合体。(3)分别以T细胞敲除小鼠(C57BL/6TCRα-KO)与正常C57BL/6小鼠作为受体,给予同样的TBI(450cGy)+BMT预处理,然后进行心脏移植。BMT后28天常规检测血中嵌合体。结果:(1)移植心脏存活时间的比较:给予ATS+亚致死量TBI(450cGy)+骨髓移植(BMT)联合预处理,效果最好,与致死量TBI(800cGy)+骨髓移植(BMT)联合预处理相比,没有显著性差异(p0.05)。而TBI(450cGy)+骨髓移植(BMT)组的移植心平均存活时间仅22.1±9.4天,与前2组对比呈显著性差异(p0.001)。其他仅给予ATS或/和TBI、ATS+BMT等预处理的对照组,其移植心脏均在早期出现排斥反应。(2)外周血中嵌合体分析:给予TBI(450/800cGy)+BMT与ATS+TBI(450cGy)+BMT预处理组,BMT后28天均完全异体细胞嵌合,嵌合率99%,它们之间没有显著性差异(p0.05)。但TBI(800cGy)+ BMT与ATS+TBI(450cGy)+BMT组的嵌合体中,供者T细胞比例明显高于TBI(450cGy)+BMT组,呈显著性差异(p0.001),说明前两组预处理方案易于使供者T细胞增殖。ATS+ +BMT(无TBI)组,术后28天未能检测出嵌合体,说明单纯使用ATS嵌合体形成不稳定,消失早。在对照组中,TBI(450cGy)预处理组,术后28天血中T细胞比例明显低于单独ATS(无TBI)预处理组,呈显著性差异(p0.01),说明TBI可以有效地杀伤受者T细胞。(3)BMT后早期(第7天):TBI(450cGy)+BMT预处理组,在外周血与脾脏中均可看到受体本身T细胞存活,在胸腺中几乎为受体自身细胞,而TBI(800cGy)+ BMT、ATS+TBI(450cGy)+BMT两组,在外周血中几乎看不到受体本身T细胞,在脾脏中仅看到极少数,在胸腺中已出现较多供者细胞嵌合。(4)予TBI(450cGy)+BMT预处理的C57BL/6TCRα-KO小鼠组,其移植心脏全部获得长期存活,BMT后28天获完全异体细胞嵌合,而给予同样预处理的C57BL/6小鼠其移植心脏存活时间短,BMT后28天嵌合体形成不稳定,其中大多数嵌合率10%。两者均呈显著性差异(p0.001)。说明:无T细胞宿主可以使供者造血干细胞在同等预处理条件下易于植活,形成较稳定的嵌合体,诱导长期的供者特异性免疫耐受。结论:(1)持久的供者造血干细胞嵌合体是诱导供-受者特异性长期稳定免疫耐受的重要条件。(2)移植早期彻底清除或灭活受体抗供体抗原的成熟T细胞,不但有利于避免早期急性排斥反应,而且有利于供者造血干细胞的植活,以获得持久稳定的异体造血干细胞嵌合体与中枢性免疫细胞克隆清除,诱导特异性长期耐受。(3)亚致死量放射治疗仍是目前用于灭活受体成熟T细胞、提高供者造血干细胞植货活、形成嵌合体最有效、经济、方便的主要手段之一。(4)本研究表明:移植前ATS+TBI(450cGy)+BMT预处理方案效果最佳,可诱导供者特异性长期免疫耐受,并无GVHD、感染、慢性排斥反应等并发症。此方案在各项治疗时间安排上比较适合于临床,可供临床应用借鉴,有重要的实用价值和指导意义。
[Abstract]:Objective: (1) By comparing various pretreatment schemes, a low toxicity, suitable for clinical trials, can induce long-term stable donor-specific immune tolerance and other advantages of the pretreatment scheme, to provide a reliable experimental basis for further clinical trials. (2) To explore the early residual host antigen in the transplantation of mature T cells. The role of acute rejection, hematopoietic stem cell proliferation and immune tolerance in order to better guide the establishment of an effective and feasible scheme for inducing immune tolerance.
Methods: (1) The model of heart transplantation was established in mice after ear transplantation. Total body X-ray irradiation (TBI), injection of anti-thymocyte serum (ATS) and infusion of donor C57BL/6 (H-2b) bone marrow cells (5.0 *10~6) were given to recipient BALB/C (H-2d) mice with different pretreatment. After heart transplantation, no immunosuppressive therapy was given. Chimerism in peripheral blood was detected routinely 28 days after bone marrow transplantation. (2) BALB/C mice were divided into three groups and given TBI (800cGy) + BMT, TBI (450cGy) + BMT, ATS + TBI (450cGy) + BMT, respectively, on the 7th, 14th, 21st, 28th and 42nd days after BMT. Chimeras in peripheral blood, spleen and thymus were measured. (3) T-cell knockout mice (C57BL/6TCR alpha-KO) and normal C57BL/6 mice were used as recipients respectively, and the same TBI (450cGy) + BMT pretreatment was given. The blood chimeras were routinely detected 28 days after BMT.
Results: (1) Comparing the survival time of transplanted heart: The combined pretreatment of ATS + sub-lethal dose TBI (450cGy) + bone marrow transplantation (BMT) was the best, and there was no significant difference (p0.05) compared with the combined pretreatment of lethal dose TBI (800cGy) + bone marrow transplantation (BMT). Chimerism analysis in peripheral blood: TBI (450/800 cGy) + BMT and ATS + TBI (450 cGy) + BMT preconditioning groups, BMT 28 days after BMT were completely allogeneic chimerism, chimerism rate was 99%. There was no significant difference between the two groups (p0.05). However, in the chimeras of TBI (800cGy) + BMT and ATS + TBI (450cGy) + BMT groups, the donor T cell ratio was significantly higher than that of TBI (450cGy) + BMT group (p0.001), indicating that the two preconditioning regimens were easy to proliferate donor T cells. TBI (450cGy) + BMT pretreatment group, 28 days after operation, the proportion of T cells in blood was significantly lower than that of ATS (without TBI) pretreatment group, showing significant difference (p0.01), indicating that TBI can effectively kill the recipient T cells. (3) Early after BMT (7th day): TBI (450cGy) + BMT pretreatment group, in peripheral blood and BMT pretreatment group. In the spleen, the T cells of the receptor itself survived, and in the thymus they were almost the recipient cells. In the TBI (800cGy) + BMT, ATS + TBI (450cGy) + BMT groups, the T cells of the receptor itself could hardly be seen in the peripheral blood, only a few in the spleen, and more donor cells were chimerized in the thymus. (4) TBI (450cGy) + BMT pretreated C57BL / BMT. In 6TCR alpha-KO mice, all the transplanted hearts survived for a long time, and complete allogeneic chimerism was obtained 28 days after BMT. However, C57BL/6 mice given the same pretreatment had a short survival time, and the chimerism formation was unstable 28 days after BMT. Most of the chimerism rates were 10%. There was a significant difference between the two groups (p0.001). Donor hematopoietic stem cells can be easily transplanted under the same pretreatment conditions to form stable chimeras and induce long-term donor-specific immune tolerance.
CONCLUSIONS: (1) Persistent donor-recipient hematopoietic stem cell chimerism is an important condition for inducing donor-recipient specific long-term stable immune tolerance. (2) Complete removal or inactivation of mature T cells from recipient antigen at early stage of transplantation is beneficial not only to avoid early acute rejection, but also to the transplantation and survival of donor-recipient hematopoietic stem cells. (3) Sublethal radiation therapy is still one of the most effective, economical and convenient methods for inactivating mature T cells of recipients, improving donor hematopoietic stem cell transplantation and forming chimeras. Pre-ATS+TBI(450cGy)+BMT preconditioning regimen has the best effect and can induce donor-specific long-term immune tolerance without GVHD, infection, chronic rejection and other complications.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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