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  • 學位論文

俱血管骨髓移植引導移植體中供體特定耐受性

Vascularized bone marrow transplantation induce donor-specific tolerance to allograft

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摘要


概論: 複合性組織異體移植像手與膝蓋移植可以被視為一種帶血管骨髓移植. 帶血管骨髓移植已被證明可以比骨髓細胞注射更快速重組淋巴血液系統,骨髓細胞來自於帶血管骨植體可以建立嵌合體,多數這些嵌合體可以建立耐受性,卻也引起植體對抗宿主疾病,我們在這裡提出帶血管骨髓移植在非骨髓移除的調控與短期的抗排斥藥物可以避免植體對抗受體疾病,引起對複合性組織異體的贈體專一性的耐受性。 材料與方法: 八到十周大雄性Brown Norway大鼠當作贈體,十到十二周大的雄性Lewis大鼠當作受體, 實驗分組如下:第一組,950 cGy全身性輻射線照射,沒有帶血管骨髓移植。第二組,950 cGy全身性輻射線照射, 同種同基因型帶血管骨髓移植。 第三組,950 cGy全身性輻射線照射, 同種異基因型帶血管骨髓移植。第四組,同種同基因型帶血管骨髓移植,沒有全身性輻射線照射及免疫調控處理。第五組,同種異基因型帶血管骨髓移植,沒有全身性輻射線照射及免疫調控處理。第六組,同種異基因型帶血管骨髓移植,600 cGy全身性輻射線照射加上免疫抗排斥藥。第一、二、三組在帶血管骨髓移植前一天進行950 cGy全身性輻射線照射,第六組在帶血管骨髓移植前一天進行腹腔內注射5mg抗淋巴球血漿與全身性輻射線照射,從0到10天腹腔內注射1m/kg的Tacrolimus ,第10天注射腹腔內注射5mg抗淋巴球血漿。所有受體在移植前,骨髓細胞在骨髓腔內被沖洗與刮除出來。移植手術後是否發生排斥與植體對抗受體疾病會藉由臨床與病理切片評估,嵌合體值與各血液細胞群的分布在移植後會用細胞流速測定儀測量。 結果:所有第一組在致死輻射線照射後在12天內死亡。同種同基因型帶血管骨髓移植與同種異基因型帶血管骨髓移植可以延長受體的生命。第三組同種異基因型後肢骨肌肉皮辦會發展出植體對抗受體疾病,所有動物會在三周左右死亡。在第三組的嵌合體值在98.85%。細胞流速測定儀測量顯示出贈體的血液幹細胞有植入與各血液細胞群有被重建出來。在第四組可以永久接受同種同基因型植體,第五組同種異基因型植體會在14天內被排斥,在第六組中,100 %有植體被完全接受,16 %發生植體對抗受體疾病,供體的各血液細胞群可以被偵測出來。 結論:在帶血管骨植體中的血液幹細胞可以建立嵌合體與在非骨髓全移除的調控下建立對複合組織異體移植的耐受性。帶血管骨髓移植最大的好處是可以直接提供骨髓良好的環境與沒有任何延遲地,同時建立對複合組織異體移植的耐受性。我們的結論是:適度的處理帶血管植體、非骨髓全移除調控、與短期的抗排斥藥如tacrolimus與淋巴球移除血清,對於建立異體移植的耐受性與減少從帶血管骨髓移植引起植體對抗宿主疾病的負作用具重要性。

並列摘要


Introduction: Composite tissue allotransplantation (CTA) such as knee and hand transplants may be regarded as vascularized bone marrow transplantation (VBMT). It has been proven that lymphohematopoietic reconstitution with VBMT was faster than bone marrow (BM) cell infusion. BM cells from the donor vascularized bone graft (VBG) may induce chimerism. The majority of these chimeras undergo tolerance while a minority develops graft versus host disease (GVHD) in rats. We report here VBMT after proper manipulation can avoid GVHD and induce donor-specific tolerance to CTA with the nonmyeloablative conditioning and short-term immunosuppressive treatment. Materials and methods. Male (8- and 10- week old) Brown Norway rats (RTA1C) were donors and male (10 – to 12-week old) Lewis rats (RTA1l) were recipients. Experimental groups were: Group I: 950 cGy total body irradiation (TBI) without VBMT. Group II: syngeneic VBMT with 950 cGy of TBI. Group III: allogeneic VBMT with 950 cGy of TBI. Group IV: syngeneic VBMT without TBI and immunomodulation. Group V: allogeneic VBMT without TBI and immunomodulation. Group VI: allogeneic VBMT conditioning with 600 cGy of TBI and immunosuppressant. Recipients in group I, II, III, VI were conditioned with TBI on day-1. Recipients in group VI were treated with 5 mg anti-lymphocyte serum (ALS) intraperitoneally (IP) and TBI on day-1, tacrolimus (1mg/kg) IP from days 0 to 10, and 5mg ALS IP on day 10. All of recipients in Group II, III, IV, V, VI were transplanted with hindlimb osteomyocutaneous (OMC) flap as VBMT on day 0. Before OMC flap transplantation, the BM cells in the bone cavity were flushed and curetted. Rejection and GvHD were assessed clinically and pathologically. Chimerism level and multilineage cells were assessed by flow cytometry after transplantation. Results. All rats in Group I died in 12 days after lethally irradiation dose. Syngeneic and allogeneic VBMT could prolong the life of the recipient rats after lethal irradiation. The allogeneic hindlimb OMC flap transplantion group (group III) developed GvHD, and all animals died around 3 weeks. The average of chimerism level in group III was 98.85%. The flow cytometry showed engraftment of the donor hematopoietic stem cell with reconstitution of multilineaged lymphohematopoietic cells. Group IV accepted the syngeneic grafts permanently. Allografts in Group V were rejected within 14 days. In group VI, the acceptance rates of allograft were 100 %, and occurrence rate of GVHD was 16 %. The donor-derived multilineage cells could be detected in the recipient blood with allograft acceptance. Conclusion. Hematopoietic stem cells (HSC) from the VBG in the hindlimb OMC flap can create chimerism and tolerance to CTA in the nonmyeloablative conditioning. The great advantage of VBG in CTA are directly offering the BM niches of HSCs and simultaneous creation of tolerance to CTA without any delay. We conclude that appropriate manipulation of VBG, partial conditioning and short-term immunosuppressants such as tacrolimus and lymphocyte-depleting serum are critical for tolerance induction of allograft and reduce the side effect of GVHD from VBMT.

參考文獻


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