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  本文選題：調(diào)節(jié)性T細胞 + 轉(zhuǎn)化生長因子�。� 參考：《華東師范大學》2013年碩士論文

【摘要】：調(diào)節(jié)性T細胞是一種具有免疫抑制功能的T細胞亞群,在免疫調(diào)節(jié)、維持外周免疫耐受及抑制移植排斥反應方面有重要作用。目前研究較多的為CD4+CD25+Foxp3+調(diào)節(jié)性T細胞(Tregs),是來源于胸腺的天然Treg細胞。由于在外周血數(shù)目較少,其應用于臨床細胞治療首先需要解決的問題就是體外大量擴增得到純度高、抑制功能強和穩(wěn)定性好的Treg細胞。目前較多的是采用白細胞介素2(IL-2)和anti-CD3/CD28單克隆抗體在體外擴增Treg細胞,加入免疫抑制劑雷帕霉素以提高擴增的純度。 本文對體外擴增培養(yǎng)Treg細胞方法做了改進,即培養(yǎng)過程中加入低濃度的轉(zhuǎn)化生長因子(TGF-β1),并探討了其對Treg細胞表型及功能的影響。采用免疫磁珠分選的方法從健康人外周血單個核細胞中分離出CD4+CD25+T細胞,用CD3/CD28單克隆抗體包被的免疫磁珠和IL-2刺激,加入雷帕霉素抑制效應T細胞擴增,此為對照組；實驗組加入3ng/ml TGF-β1,培養(yǎng)十二天收獲Treg檢測表型及功能。實驗結果顯示,TGF-β1可以顯著上調(diào)HLA-DR以及Helios的表達,也提高了Treg細胞其它標志及功能分子的表達,如Foxp3, CTLA-4,LAP等；體外擴增的Treg細胞不僅對自體的效應T細胞有抑制作用,同時對異體的效應T細胞也有抑制作用,且TGF-β1增強了實驗組Treg細胞的免疫抑制功能；TGF-β1提高了Treg細胞的純度,實驗組分泌更少的IL-4、IL17、IFN-Y。 本文還構建了一個aGVHD-SCID小鼠模型,進一步驗證了TGF-β1可以提高體外擴增Treg細胞的免疫抑制功能. SCID小鼠經(jīng)半致死劑量光照,24小時后腹腔注射人外周血單個核細胞(PBMC)誘導aGVHD,實驗組輸注體外擴增的Treg細胞進行治療。后續(xù)觀察發(fā)現(xiàn),小鼠相繼出現(xiàn)弓背、體重下降、脫毛等GVHD癥狀,且生存時間比單光照小鼠顯著縮短；經(jīng)人Treg細胞治療,癥狀明顯減輕,生存期延長,且TGF-β1處理的Treg細胞治療效果更好。 有文獻報道HLA-DR+Treg細胞比HLA-DR-Treg細胞有更強的免疫抑制功能,本實驗首次發(fā)現(xiàn)TGF-β1顯著上調(diào)了Treg細胞HLA-DR的表達,而其它文獻報道TGF-β1通過抑制IFN-γ信號通路下調(diào)間充質(zhì)干細胞、星形膠質(zhì)細胞瘤中HLA-DR的表達。因此,本文還初步探討了TGF-β1上調(diào)Treg細胞表達HLA-DR可能的機制。
[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- 緯 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.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R392.11

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本文編號：1807491