摘要
目的建立在体外从健康成人外周血单核细胞(Mo)诱导培养成熟的树突状细胞(DC)的方法。方法采用密度梯度离心法分离健康成人外周血中的单个核细胞(PBMC),再以黏附法分离出Mo,加入重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)体外培养12天,并于培养第6天加入重组人肿瘤坏死因子(rhTNF-α)共同培养,于倒置显微镜下观察细胞的形态,以流式细胞仪检测培养6、9、12天DC表面CD1a、CD83、CD86和MHC-DR的表达水平,用MTT法测定DC刺激同种异体T细胞增殖的能力,进行比较分析。结果Mo经rhGM-CSF+rhIL-4诱导培养6 d后,细胞成簇,表型为CD1a 53.2%、CD83 13.6%、CD86 65.5%、MHC-DR 75.4%;TNF-α诱导后,即培养第9 d,细胞表型为CD1a 62.1%、CD83 73.5%、CD86 92.3%、MHC-DR 98.4%,激发初始T细胞增殖的能力最强。结论rhGM-CSF+rhIL-4诱导Mo 6 d,可获得大量不成熟的DC,该体系有利于DC扩增;加入TNF-α,培养第9 d,DC成熟度最高,适合于临床免疫治疗。
Objective To find a way by which dendritic cells （DC） from healthy human monocyte in peripheral blood might be induced to be matured. Methods Mononuclear cells were isolated from peripheral blood of healthy human by Ficoll-Hypaque density gradient separation, and then monocytes（Mo） were purified through adhensiveness. DCs were obtained after a 12 day incubation of monocytes with the media containing granulo-cyte/macrophage colony stimulating factor （GM-CSF）, interleukin-4 （IL-4） and tumor necrosis factor-α（TNF-α） which was added on the 6th day. DC＇s morphology was observed under inverted microscope, the expressions of CDla, CD83, CD86 and MHC-DR on DCs were analyzed by flow cytometer on the 6th,9th, 12th day, and the abilities to stimulate the proliferation of allogenic T cells by DCs were evaluated by MTT assay. Results After a 6 day incubation of monocytes with GM-CSF and IL-4, most of them formed clusters, and the expression levels of CD1a, CD83, CD86 and MHC-DR were 53.2%, 13.6%, 65.5 %, 75.4%, respectively. On the 9th day in the presence of TNF-α, the expression levels of CDla, CD83, CD86 and MHC-DR of DCs were 62.1%, 73.5 % ,92.3%, 98.4%, respectively, and the proliferation of native T cells induced by DCs was best enhanced. Conclusion It is suggested that combination of GM-CSF and IL-4 is beneficial to expanding and in- ducing immature DCs, and the matured DCs best induced by adding TNF-α into the media on the 9th day can be used for clinical immunotherapy.
出处
《哈尔滨医科大学学报》
CAS
北大核心
2008年第3期243-245,250,共4页
Journal of Harbin Medical University
基金
广东省自然科学基金(07300964)
