供者未成熟树突状细胞防治移植物抗宿主病的实验研究

发布时间：2018-07-03 18:10

本文选题：树突状细胞 + 阻断疗法　；参考：《石河子大学》2008年硕士论文

【摘要】： 目的:依据GVHD发生机制构建体外DC诱导T淋巴细胞的调节模型,观察供者未成熟树突状细胞(immature dendritic cell,imDC)刺激白体T细胞增殖的能力,探讨利用imDC治移植物抗宿主病(GVHD)临床应用的可行性。方法:从健康供者外周血分离单核细胞,采用重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素(IL)-4联合培养5天,诱导其分化成imDC;诱导培养7天,加脂多糖(LPS)刺激18小时后,分化成熟树突状细胞(mature dendritic cell,mDC)。并通过倒置显微镜和HE染色观察细胞形态,细胞滴片免疫组化检测mDC表面MHC-II类分子HLA-DR、CD86的表达情况.利用流式细胞仪检测imDC和mDC表面DC特异性分子标记CDla和成熟标记CD83的阳性表达率。采用单向MLR方法,进行供受者混合淋巴细胞培养,构建DC诱导T淋巴细胞的调节模型,将实验设为3组:对照组、imDC组和mDC组,共孵育48小时后,加入CCK-8,用酶标仪检测各组OD值,比较供者imDC和mDC刺激自体T细胞增殖的能力。结果:(1)培养5天后细胞呈半悬浮状生长,周边毛刺较粗短,形态不一,细胞体积较单核细胞增大具有典型的imDC形态学特征,流式细胞仪检测CDla、CD83和双抗分别表达为(67.06±0.93)%、(66.82±5.06)%和(62.34±1.94)%;培养8天后细胞呈单个悬浮状生长,胞体圆形,体积较大,细胞周边可见明显较长突起,具有典型mDC形态学特征,细胞滴片免疫组化结果显示HLA-DR、CD86阳性,流式细胞仪检测CDla、CD83和双抗表达分别为(64.98±2.99)%、(86.44±4.10)%和(65.16±0.55)%。(2)单向MLR法混台淋巴细胞培养共孵育48小时后,加入CCK-8检测OD值,imDC组与对照组比较无统计学意义(P0.05):mDC组与对照组、imDC组比较均有显著统计学意义(P0.01),对自体T细胞的刺激指数大于2.00(SI=2.22),与2.00相比差异有统计学意义(P0.05)。结论:形态学观察、免疫组化及流式细胞仪细胞表型检测结果提示,rhGM-CSF+IL-4联合成功诱导培养出了imDC和mDC;在单向MLR法构建的DC诱导T淋巴细胞的调节模型中CCK-8法检测结果提示imDC可以诱导白体T细胞低应答,有可能成为防治GVHD的一种方法。
[Abstract]:Aim: to establish a regulatory model of T lymphocytes induced by DC in vitro according to the mechanism of GVHD, and to observe the ability of donor immature dendritic cells (immature dendritic cells) to stimulate the proliferation of leukocytes. To explore the feasibility of clinical application of IMDC in the treatment of graft-versus host disease (GVHD). Methods: mononuclear cells were isolated from the peripheral blood of healthy donors and co-cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and interleukin-4 (IL-4) for 5 days to induce their differentiation into imDC.After 7 days of induction and 18 hours of lipopolysaccharide (LPS) stimulation, Differentiation of mature dendritic cells (mature dendritic cell / mDC). The cell morphology was observed by inverted microscope and HE staining. The expression of MHC-II molecule HLA-DRN CD86 on MDC was detected by immunohistochemistry. The positive rates of DC specific molecular marker CDla and mature marker CD83 on the surface of imDC and mDC were detected by flow cytometry. Single MLR method was used to culture donor mixed lymphocytes, and the regulatory model of T lymphocytes induced by DC was established. The experiment was divided into three groups: control group and mDC group. After incubation for 48 hours, CCK-8 was added to each group. OD value of each group was detected by enzyme marker. To compare the ability of donor imDC and mDC to stimulate the proliferation of autologous T cells. Results: (1) after 5 days of culture, the cells grew semi-suspended, the peripheral burr was shorter and thicker, the morphology was different, and the cell volume was larger than that of monocytes, which had typical morphological characteristics of imDC. The expression of CD83 and double antibody were (67.06 卤0.93), (66.82 卤5.06)% and (62.34 卤1.94), respectively. The expression of CD83 and double antibody were (64.98 卤2.99)%, (86.44 卤4.10)% and (65.16 卤0.55)%, respectively. (2) the expression of CD83 and double antibody were (64.98 卤2.99), (86.44 卤4.10)% and (65.16 卤0.55), respectively. There was no significant difference in OD value between the control group and the control group (P0.05). The stimulation index of autologous T cells was more than 2.00 (SI-2.22) in the control group (P0.01), and the difference was significant compared with the control group (P0.05) (P0.05). Conclusion: morphological observation, The results of immunohistochemistry and flow cytometry showed that rhGM-CSF IL-4 combined with rhGM-CSF IL-4 could induce imDC and mDC.The CCK-8 method showed that imDC could induce T lymphocytes in the model of DC induced by unidirectional MLR. White body T cell low response, It may be a method to prevent GVHD.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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