非清髓性骨髓移植诱导异基因受者小鼠免疫耐受的实验研究

Induction of Immune Tolerance for Allogenic Recipient Mice by Non-Myeloablative Bone Marrow Transplantation

本研究通过非清髓性预处理方案联合髓腔内骨髓移植(IBMBMT)建立异基因小鼠免疫耐受模型,并探讨其诱导耐受的机理。受鼠为雌性C57BL/6(H2b,B6)小鼠,于第0天接受60Coγ线全身照射(TBI),4小时内输注雄性BALB/c(H2d)小鼠来源的骨髓细胞(BMC),2天后腹腔注射环磷酰胺(CTX)。通过皮肤移植、混合淋巴细胞反应(MLR)检测耐受状态,并通过体外过继转移实验、IL2逆转实验等探讨免疫耐受的机制。结果显示,经骨髓移植的B6小鼠对BALB/c小鼠的皮肤移植物平均存活时间(MST)>150天,较对照组明显延长(P<0.01);骨髓移植后第90天,受鼠(黑色)表型开始呈现供鼠(白色)颜色特征。MLR结果证明,B6小鼠获得供体特异性耐受,该耐受可以被IL2逆转且可被过继转移;所有受鼠均未出现GVHD表现。结论:非清髓预处理联合髓腔内骨髓移植可以有效地诱导异基因小鼠免疫耐受,克隆无能、抑制细胞存在及嵌合体产生均参与耐受的形成。

This study was aimed to explore the effects and mechanisms of transplantation tolerance induced by “TBI ＋ cyclophosphamide （CTX）” regimen combined with intra-bone marrow injection of allogenic BMCs. On day 0 C57BL/6 （H-2^b, B6 ） mice received sublethal dose of total body irradiation （TBI） （^60Co γ-ray） followed by.intrabone marrow-bone marrow transplatation （IBM-BMT） of 3 × 10^7cells/30μl BMCs from BALB/c （H-2^d） mice. The recipient mice were given CTX intraperitoneally 2 days after IBM-BMT. On day 7 skin grafting was performed and the skin survival was observed. The tolerance mechanism was investigated by mixed lymphocyte reaction （ MLR）, IL-2 reverse test, adoptive transfer assay in vitro. The results showed that the mean survival time （MST） of skin allografts in group treated with TBI ＋ CTX ＋ BMT was significantly longer, compared with that of other groups （P 〈 0.01 ）. On day 90 after IBM-BMT, the phenotypic character of the recipient mice （black color） began to convert to that of the donor mice （white color）. The MLR demonstrated that the immune responses of recipient mice were donor-specific tolerance. Suppressive activity in the spleen cells of tolerant B6 mice was observed in adoptive transfer assay in vitro. IL-2 reversal and the phenotypic conversion showed that the tolerance mechanisms were involved in clonal anergy and the development of chimerism. It is concluded that the nonmyeloablative regimen combined with IBM-BMT can induce a long-term tolerance, and the multiple mechanisms including clonal anergy, suppressor cells and chimerism were involved in transplantation immune tolerance.