转化生长因子对体外扩增调节性T细胞HLA-DR表达的影响

发布时间：2018-04-26 20:14

本文选题：调节性T细胞 + 转化生长因子　；参考：《华东师范大学》2013年硕士论文

【摘要】：调节性T细胞是一种具有免疫抑制功能的T细胞亚群,在免疫调节、维持外周免疫耐受及抑制移植排斥反应方面有重要作用。目前研究较多的为CD4+CD25+Foxp3+调节性T细胞(Tregs),是来源于胸腺的天然Treg细胞。由于在外周血数目较少,其应用于临床细胞治疗首先需要解决的问题就是体外大量扩增得到纯度高、抑制功能强和稳定性好的Treg细胞。目前较多的是采用白细胞介素2(IL-2)和anti-CD3/CD28单克隆抗体在体外扩增Treg细胞,加入免疫抑制剂雷帕霉素以提高扩增的纯度。本文对体外扩增培养Treg细胞方法做了改进,即培养过程中加入低浓度的转化生长因子(TGF-β1),并探讨了其对Treg细胞表型及功能的影响。采用免疫磁珠分选的方法从健康人外周血单个核细胞中分离出CD4+CD25+T细胞,用CD3/CD28单克隆抗体包被的免疫磁珠和IL-2刺激,加入雷帕霉素抑制效应T细胞扩增,此为对照组；实验组加入3ng/ml TGF-β1,培养十二天收获Treg检测表型及功能。实验结果显示,TGF-β1可以显著上调HLA-DR以及Helios的表达,也提高了Treg细胞其它标志及功能分子的表达,如Foxp3, CTLA-4,LAP等；体外扩增的Treg细胞不仅对自体的效应T细胞有抑制作用,同时对异体的效应T细胞也有抑制作用,且TGF-β1增强了实验组Treg细胞的免疫抑制功能；TGF-β1提高了Treg细胞的纯度,实验组分泌更少的IL-4、IL17、IFN-Y。本文还构建了一个aGVHD-SCID小鼠模型,进一步验证了TGF-β1可以提高体外扩增Treg细胞的免疫抑制功能. SCID小鼠经半致死剂量光照,24小时后腹腔注射人外周血单个核细胞(PBMC)诱导aGVHD,实验组输注体外扩增的Treg细胞进行治疗。后续观察发现,小鼠相继出现弓背、体重下降、脱毛等GVHD症状,且生存时间比单光照小鼠显著缩短；经人Treg细胞治疗,症状明显减轻,生存期延长,且TGF-β1处理的Treg细胞治疗效果更好。有文献报道HLA-DR+Treg细胞比HLA-DR-Treg细胞有更强的免疫抑制功能,本实验首次发现TGF-β1显著上调了Treg细胞HLA-DR的表达,而其它文献报道TGF-β1通过抑制IFN-γ信号通路下调间充质干细胞、星形胶质细胞瘤中HLA-DR的表达。因此,本文还初步探讨了TGF-β1上调Treg细胞表达HLA-DR可能的机制。
[Abstract]:Regulatory T cells are a subgroup of T cells with immunosuppressive function, which play an important role in immune regulation, maintenance of peripheral immune tolerance and suppression of transplantation rejection. At present, CD4 CD25 Foxp3 regulatory T cell Tregsna is a natural Treg cell derived from thymus. Due to the small number of peripheral blood, the first problem to be solved in clinical cell therapy is to obtain Treg cells with high purity, strong inhibition function and good stability by mass expansion in vitro. At present, interleukin-2 (IL-2) and anti-CD3/CD28 monoclonal antibody were used to amplify Treg cells in vitro, and to increase the purity of amplification by adding rapamycin, an immunosuppressant. In this paper, the method of in vitro amplification and culture of Treg cells was improved, that is, the low concentration of TGF- 尾 1 was added into the culture process, and the effects of TGF- 尾 1 on the phenotype and function of Treg cells were discussed. CD4 CD25 T cells were isolated from the peripheral blood mononuclear cells of healthy people by immunomagnetic bead sorting. Immunomagnetic beads and IL-2 stimulated by CD3/CD28 monoclonal antibody were used to amplify the T cells with rapamycin inhibitory effect. 3ng/ml TGF- 尾 1 was added to the experimental group and Treg was harvested for phenotype and function. The results showed that TGF- 尾 1 could significantly up-regulate the expression of HLA-DR and Helios, and also enhance the expression of other markers and functional molecules in Treg cells, such as Foxp3, CTLA-4, etc. In vitro, Treg cells not only inhibited autologous effector T cells, but also increased the expression of TGF- 尾 1. TGF- 尾 1 enhanced the immunosuppressive function of Treg cells in experimental group. TGF- 尾 1 increased the purity of Treg cells. A aGVHD-SCID mouse model was constructed, which further demonstrated that TGF- 尾 1 could enhance the immunosuppressive function of Treg cells in vitro. SCID mice were induced by human peripheral blood mononuclear cells (PBMC) 24 hours after half-lethal dose illumination. The experimental group was treated with expanded Treg cells in vitro. The follow-up observation showed that GVHD symptoms such as bow back, weight loss and hair loss appeared in mice, and the survival time was significantly shorter than that in single illumination mice. After treatment with human Treg cells, the symptoms were obviously alleviated, and the survival time was prolonged. The Treg cells treated with TGF- 尾 1 were more effective than those treated with TGF- 尾 1. It has been reported that HLA-DR Treg cells have stronger immunosuppressive function than HLA-DR-Treg cells. TGF- 尾 1 significantly up-regulated the expression of HLA-DR in Treg cells for the first time, while other literatures reported that TGF- 尾 1 down-regulated mesenchymal stem cells by inhibiting IFN- 纬 signaling pathway. Expression of HLA-DR in astrocytomas. Therefore, the possible mechanism of TGF- 尾 1 upregulation of HLA-DR expression in Treg cells was also discussed.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392.11

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