同种异体反应性细胞与调节性T细胞在新生期诱导的小鼠移植耐受中的作用探讨

摘要

Objective:To investigate the role of alloreactive T cell in clonal deletion and regulatory T cells ( Treg) in transplant tolerance.Methods:F1 mice were bred by crossing female BALB/c mice and male C57BL/6 mice.Within 24 h,newborn C57BL/6 mice were inoculated with F1 spleen cells via the orbital branch of the anterior facial vein.Six weeks later,the mice were subjected to F1 skin grafting to evaluate their tolerance.Proliferation,flow cytometry and adoptive transfer assay were used to analyze clonal deletion of alloreactive T cells and the expression of CD4+Foxp3+T cells in neonatal treated mice.Results: Newborn C57BL/6 mice injected with F1 splenic cells could induce transplantation tolerance,the level of tolerance was associated with the dose of splenic cells.3×107 splenic cells from F1 mice could induce long-term skin graft acceptance in C57BL/6 mice ,1×107splenic cells significantly prolonged the survival of F1 skin grafts,but the grafts completely rejected within 50 days.The mixed lymphocyte reaction ( MLR) experiment in vivo showed that alloreactive T cell in long-term tolerant mice was deleted completely,but a certain amount of reactive T cells existed in the low-dose group mice.Flow cytometry ( FCM) analysis showed that the expression of CD4+Foxp3+T cell in the high-dose group and low-dose group mice had no obvious difference compared with the naive mice.When alloreactive T-cells were injected into tolerant mice,the skin graft rejection was observed,and Treg cells upregulated in graft-rejected mice.Conclusion:The degree of transplantation tolerance depended on the clonal deletion of alloreactive T cells,instead of on the expression of CD4+Foxp3+Treg cells.CD4+Foxp3+regulatory T cells upregulated in graft rejected mice,which may be served as a negative feedback mechanism to control the intensity of rejection.%目的：初步探讨同种异体反应性T细胞克隆清除与调节性T细胞在小鼠移植耐受中的作用。方法：雌性BALB／c小鼠与雄性C57BL／6小鼠杂交一代获得F1小鼠，不同剂量的F1小鼠脾脏细胞经眶静脉输注给新生24 h C57BL／6小鼠体内诱导耐受，成年后移植F1小鼠来源的皮肤，建立不同耐受程度的小鼠移植耐受模型；耐受小鼠脾脏细胞经CFSE标记后注射到F1小鼠体内，分析耐受小鼠来源的T细胞在体内对F1抗原的增殖能力；流式细胞术、过继转移实验分析CD4＋Foxp3＋调节性T细胞在移植耐受和移植排斥过程中的表达。结果：C57BL／6小鼠在新生期输注F1小鼠脾脏细胞可诱导移植耐受，耐受程度与输注的脾脏细胞剂量有关，3×107个F1小鼠脾脏细胞可诱导C57BL／76小鼠长期皮肤移植耐受，1×107个细胞诱导可使移植皮肤生存时间显著延长，但在50 d内完全排斥；体内混合淋巴细胞反应实验证明，长期耐受小鼠体内的同种异体反应性T细胞被完全克隆清除，但低剂量组小鼠体内仍存在一定数量的反应性T细胞；流式细胞分析发现，高剂量和低剂量组小鼠体内的CD4＋Foxp3＋调节性T细胞表达与初始小鼠相比没有显著差异；同种异体反应性T细胞过继转移给耐受小鼠，移植耐受的皮肤发生排斥反应，小鼠体内的调节性T细胞表达升高。结论：小鼠的移植耐受程度与小鼠体内的同种异体反应性T细胞的克隆清除程度有关，与CD4＋Foxp3＋调节性T细胞的表达没有直接关系；调节细胞在移植排斥过程中表达升高，可能作为一种反馈机制参与耐受的形成。