未成熟树突状细胞防治移植物抗宿主病的实验研究

Research on Prevention and Cure Graft Versus Host Disease by Immature Dendritic Cells

目的:观察供者未成熟树突状细胞(imDC)刺激自体T细胞增殖的能力,探讨利用imDC防治移植物抗宿主病(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4d,诱导其分化成imDC;培养7d,分化成mDC。并通过倒置显微镜和HE染色观察细胞形态、流式细胞仪检测细胞表型。采用MLR方法,构建GVHD发生机制的模型,比较供者imDC和mDC刺激自体T细胞增殖的能力。结果:(1)培养4天后细胞具有典型的imDC特征,CD1a、CD83和双抗分别表达为55.79%、64.67%和46.67%,成熟标志CD83表达较低;培养7天后具有典型mDC特征,CD1a、CD83和双抗表达分别为61.56%、82.40%和64.12%,成熟标志CD83表达较高。(2)MLR法共孵育72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(P>0.05),不能刺激自体T细胞增殖(SI<1.00);mDC组与对照组、imDC组比较均有显著统计学意义(P<0.01),能够刺激自体T细胞增殖(SI>2.00)。结论:供者imDC能够诱导自体T细胞低反应,有望用来防治GVHD。

Objective： To observe the ability of immature dendritic cells that induce the stimulation of autoallergic naive T lymphocyte proliferation, and investigate the feasibility of immature dendritic cells prevention and cure GVHD in clinical application. Methods： Mononuclear cells were isolated from the peripheral blood of the healthy donor. They were induced and differentiated into immature dendritic cells with rhGM-CSF and IL-4 for 4 days, and were induced and differentiated into mature dendritic cells for 7 days. Then the morphology of the cells were examined by inverted microscope and hematoxylin and eosin stain. Immunological phenotype were examined with flow cytometry. According to the method of mixed lymphocyte reaction （MLR）, constructed the model of the generated mechanism for GVHD, to compare with the ability that induced the stimulation ofautoallergic narve T lymphocyte proliferation between donor immature and mature dendretic cells. Results： （ 1 ） The cells exhibited the typical morphology ofimDC after 4 days, each of the specific expression of CDla, CD83 and double antibody was 55.79 %, 64.67 % and 46.67 %, with lower mature expression CD83; and the cells exhibited the typical morphology ofmDC after 7 days, each of the specific expression of CDla, CD83 and double antibody was 61.56 %, 82.40 % and 64.12 %,with higher mature expression CD83; （2） dendritic cells and MLR have co-cultured for 72 hours, and doped cell counting kit-8（CCK-8 ） to detect optical density, Statistical analysis showed that there was no obvious difference between imDC and control group, which couldn＇t stimulate autoallergic T cells proliferation （S〈 1.00）. However, mDC group had significant difference between imDC and control group （P〈0.01 ）, which was capable to stimulate T lymphocyte proliferation （SI〉2.00）. Conclution： Donor imDC is capable to induce that autoallergic T lymphocyte have low reaction, and it may be used to prevent and cure graft versus host disease.