本文選題：抗原識(shí)別 + 調(diào)節(jié)性T細(xì)胞�。� 參考：《第三軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 目的CD4+CD25+調(diào)節(jié)性T細(xì)胞(Regulatory T cell,Treg)是CD4+ T細(xì)胞的一個(gè)重要亞群,具有免疫抑制性和誘導(dǎo)免疫無能兩大功能特征。其作用機(jī)制可能是通過細(xì)胞間直接接觸和/或分泌抑制性細(xì)胞因子發(fā)揮作用。在維持機(jī)體免疫內(nèi)環(huán)境穩(wěn)定、防止自身免疫性疾病發(fā)生、誘導(dǎo)移植耐受等方面都起著重要作用。但目前研究多著重于不同細(xì)胞因子的組合對(duì)Treg的影響,而忽略了不同抗原識(shí)別方式在Teg誘導(dǎo)中的重要作用。因此,本實(shí)驗(yàn)擬探討不同抗原識(shí)別方式對(duì)同種異型Treg免疫學(xué)特性的影響,求證間接識(shí)別方式在對(duì)同種異體抗原特異性Treg的生成中是否存在關(guān)鍵作用。 方法取移植前受者外周血單個(gè)核細(xì)胞(periphery blood mononuclear cell,PBMC),通過磁珠分離獲得CD4+CD25-Tcell。同時(shí)取供者PBMC于含重組人粒系-巨噬細(xì)胞系集落刺激因子(rhgranulocyte-macrophage clone-stimulatin factor,rhGM-CSF) 75ng/ml、重組人白介素4(rhinterleukin4,rhIL-4)25ng/ml和雷帕霉素(rapamycin,RAPA)20ng/ml的完全培養(yǎng)基中常規(guī)培養(yǎng)5d。收集培養(yǎng)4d后的受者未成熟樹突狀細(xì)胞(immature dendritic cell,imDC)并加入供者PBMC裂解液以使擴(kuò)增的DC負(fù)載供者抗原共培養(yǎng)3d,作為抗原提呈細(xì)胞(antigen presenting cell,APC)。再將分離純化的CD4+CD25-T細(xì)胞和負(fù)載供者抗原的受者樹突狀細(xì)胞共培養(yǎng)5d;同時(shí)加入IL-2 10ng/ml、RAPA 20ng/ml促進(jìn)細(xì)胞增殖并誘導(dǎo)特異性Treg生成,以Treg1表示。同時(shí)在Treg誘導(dǎo)生成時(shí)不加入負(fù)載供者抗原的受者樹突狀細(xì)胞,而直接采用供者PBMC作為APC,通過直接抗原識(shí)別方式生成特異性Treg,以Treg2表示。并比較2種方式生成的Treg的免疫特性及誘導(dǎo)過程中細(xì)胞因子IL-2的分泌。 結(jié)果 1.通過間接抗原識(shí)別方式生成的Treg1生成率為62.4%,其增殖抑制率為63.5%;直接抗原識(shí)別方式生成的Treg2生成率44.4%,其增殖抑制率為45.8%。 2.間接抗原識(shí)別方式誘導(dǎo)過程中細(xì)胞因子IL-2的分泌也明顯優(yōu)于直接抗原識(shí)別方式。 結(jié)論 1.與直接抗原識(shí)別方式相比,間接抗原識(shí)別方式誘導(dǎo)生成的Treg的數(shù)量更多及免疫特性更強(qiáng)。 2.間接抗原識(shí)別方式較直接抗原識(shí)別方式在誘導(dǎo)過程中能產(chǎn)生更適量的IL-2以誘導(dǎo)抗原特異性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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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