不同抗原识别方式对同种异体抗原特异性Treg免疫特性的影响

发布时间：2018-05-30 07:21

本文选题：抗原识别 + 调节性T细胞　；参考：《第三军医大学》2010年硕士论文

【摘要】： 目的CD4+CD25+调节性T细胞(Regulatory T cell,Treg)是CD4+ T细胞的一个重要亚群,具有免疫抑制性和诱导免疫无能两大功能特征。其作用机制可能是通过细胞间直接接触和/或分泌抑制性细胞因子发挥作用。在维持机体免疫内环境稳定、防止自身免疫性疾病发生、诱导移植耐受等方面都起着重要作用。但目前研究多着重于不同细胞因子的组合对Treg的影响,而忽略了不同抗原识别方式在Teg诱导中的重要作用。因此,本实验拟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对同种异体抗原特异性Treg的生成中是否存在关键作用。方法取移植前受者外周血单个核细胞(periphery blood mononuclear cell,PBMC),通过磁珠分离获得CD4+CD25-Tcell。同时取供者PBMC于含重组人粒系-巨噬细胞系集落刺激因子(rhgranulocyte-macrophage clone-stimulatin factor,rhGM-CSF) 75ng/ml、重组人白介素4(rhinterleukin4,rhIL-4)25ng/ml和雷帕霉素(rapamycin,RAPA)20ng/ml的完全培养基中常规培养5d。收集培养4d后的受者未成熟树突状细胞(immature dendritic cell,imDC)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,作为抗原提呈细胞(antigen presenting cell,APC)。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、RAPA 20ng/ml促进细胞增殖并诱导特异性Treg生成,以Treg1表示。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特异性Treg,以Treg2表示。并比较2种方式生成的Treg的免疫特性及诱导过程中细胞因子IL-2的分泌。结果 1.通过间接抗原识别方式生成的Treg1生成率为62.4%,其增殖抑制率为63.5%;直接抗原识别方式生成的Treg2生成率44.4%,其增殖抑制率为45.8%。 2.间接抗原识别方式诱导过程中细胞因子IL-2的分泌也明显优于直接抗原识别方式。结论 1.与直接抗原识别方式相比,间接抗原识别方式诱导生成的Treg的数量更多及免疫特性更强。 2.间接抗原识别方式较直接抗原识别方式在诱导过程中能产生更适量的IL-2以诱导抗原特异性Treg的生成。
[Abstract]:Objective CD4 CD25 regulatory T cell tregis is an important subgroup of CD4 T cells, which is characterized by immunosuppressive and induced immune incompetence. The mechanism may be through direct contact between cells and / or secretion of inhibitory cytokines. It plays an important role in maintaining the internal immune environment, preventing autoimmune diseases and inducing transplantation tolerance. However, many studies have focused on the effect of different cytokines on Treg, and neglected the important role of different antigen recognition methods in Teg induction. Therefore, this experiment aims to explore the effect of different antigen recognition methods on the immunological characteristics of allogeneic Treg, and to verify whether indirect recognition mode plays a key role in the generation of allogeneic antigen-specific Treg. Methods Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood mononuclear cells (PBMC) of recipients before transplantation and CD4 CD25-T cells were isolated by magnetic beads. At the same time, donor PBMC was cultured in the complete culture medium containing recombinant human granulocyte-macrophage clone-stimulatin factor-rhGM-CSF75 ng / ml, recombinant human interleukin 4, rhIL-4, 25 ng / ml and rapamycin RAPA 20 ng / ml for 5 days, at the same time, the donor PBMC was cultured in the complete culture medium containing rhgranulocyte-macrophage clone-stimulatin factor-rhGM-CSF75 ng / ml, rhIL-4 and rapamycin RAPA20ng/ ml for 5 days. Immature dendritic cells were collected from immature dendritic cells of recipients after 4 days of culture and donor PBMC lysate was added to make the expanded DC loaded with donor antigen for 3 days. The antigen was presented as antigen presenting cells of presenting cells. The purified CD4 CD25-T cells and dendritic cells loaded with donor antigen were co-cultured for 5 days, and IL-2 10 ng / ml rrapa 20ng/ml was added to promote cell proliferation and induce specific Treg production, as indicated by Treg1. At the same time, the recipient dendritic cells loaded with donor antigen were not added to Treg induction, and donor PBMC was directly used as PBMC, and specific Tregs were generated by direct antigen recognition, expressed as Treg2. The immune characteristics of Treg and the secretion of cytokine IL-2 during induction were compared. Result 1. The formation rate of Treg1 by indirect antigen recognition was 62.4, the inhibition rate of proliferation was 63.5, and the rate of Treg2 generated by direct antigen recognition was 44.4, and the inhibition rate of Treg2 proliferation was 45.8. 2. The secretion of cytokine IL-2 in the induction of indirect antigen recognition was also superior to that of direct antigen recognition. Conclusion 1. Compared with direct antigen recognition, indirect antigen recognition induces more Treg and stronger immune characteristics. 2. Indirect antigen recognition can induce antigen-specific Treg by producing more appropriate IL-2 during induction than direct antigen recognition.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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