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目的探讨不同抗原识别方式对同种异型Treg免疫学特性的影响,求证间接识别方式在特异性Treg的生成中是否存在关键作用。方法取移植前受者PMBC,通过磁珠分离获得CD4+CD25-T细胞。同时取供者PB-MC于含rhGM-CSF75ng/ml、rhIL-425ng/ml和雷帕霉素(20ng/ml)的完全培养基中常规培养5d。收集培养4d后的树突状细胞并加入供者PBMC裂解液以使扩增的DC负载供者抗原共培养3d。再将分离纯化的CD4+CD25-T细胞和负载供者抗原的受者树突状细胞共培养5d;同时加入IL-2(10ng/ml)、雷帕霉素(20ng/ml)促进细胞增殖并诱导Treg1生成。同时在Treg诱导生成时不加入负载供者抗原的受者树突状细胞,而直接采用供者PBMC作为APC,通过直接抗原识别方式生成Treg2。并比较2种方式生成的Treg的免疫特性。结果通过间接抗原识别方式生成的Treg1其增殖抑制率为63.5%,优于45.8%的直接抗原识别方式生成的Treg2。结论与直接抗原识别方式相比,间接抗原识别机制可产生更过量、特异性免疫抑制作用更强的Treg,天然Treg不能满足移植术后诱导免疫耐受的需要。
Objective To investigate the effect of different antigen recognition methods on the immunological characteristics of allo-Tregs and to confirm whether there is a key role of indirect recognition in the generation of specific Tregs. Methods PMBC was harvested before transplantation and CD4 + CD25-T cells were isolated by magnetic beads. Donor PB-MC was also routinely cultured in complete medium containing rhGM-CSF 75 ng / ml, rhIL-425 ng / ml and rapamycin (20 ng / ml) for 5 days. Dendritic cells after 4 days of culture were collected and donor PBMC lysate was added to co-culture the expanded DC-loaded donor antigen for 3 days. The purified CD4 + CD25-T cells were co-cultured with the recipient dendritic cells loaded with donor antigen for 5 days. Meanwhile, IL-2 (10ng / ml) and rapamycin (20ng / ml) And induce Treg1 production. At the same time, dendritic cells loaded with donor antigen were not added in the induction of Treg production, but donor PBMC was directly used as APC to generate Treg2 by direct antigen recognition. The immune characteristics of Tregs generated by the two methods were compared. Results The proliferation of Treg1 induced by indirect antigen recognition was 63.5%, better than that of Treg2 generated by direct antigen recognition of 45.8%. Conclusion Compared with direct antigen recognition, indirect antigen recognition can produce more excessive and specific immunosuppressive effects of Treg, natural Treg can not meet the needs of induced immune tolerance after transplantation.