鸭骨髓源树突状细胞的诱导培养及功能分析

Induced cultivation and functional analysis of duck bone marrow-derived dendritic cells

旨在建立鸭骨髓源树突状细胞(DC)体外诱导培养方法,分析DC基本生物学功能。在无菌条件下分离鸭骨髓单核细胞,优化粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)工作浓度,在体外进行诱导培养,然后通过形态学观察、吞噬活性检测、表面标志物流式鉴定、分泌细胞因子和刺激淋巴细胞增殖能力的测定,对制备的DC进行形态和生物功能评价。结果:添加20 ng/mL的IL-4和40 ng/mL的GM-CSF可以诱导出高吞噬活性的DC,诱导培养后3 d可见菱形、有纤突的细胞;经脂多糖(LPS)刺激成熟的DC,呈现典型的树突状结构,细胞表面CD11c、CD80和MHCⅡ分子表达量分别为80.08%、81.27%和91.47%;DC培养体系中白细胞介素2(IL-2)、γ干扰素(IFN-γ)分泌量随着培养时间延长而增加,并在成熟后分泌量显著升高(P<0.05);CCK-8法检测结果显示成熟后的DC以1∶5比例与淋巴细胞混合培养,能够显著刺激淋巴细胞增殖。本试验成功建立鸭骨髓源DC体外诱导培养方法,制备细胞具备了树突状细胞的基本生物学功能。

To establish a method for inducing cultivation of duck bone marrow-derived dendritic cells(DC)in vitro in order to analyze the basic biological functions of DC,duck bone marrow monocytes were isolated under sterile conditions,and the working concentration of GMCSF and IL-4 was optimized so that to co-induce cultivation of DC.Then,the prepared DC underwent a series of identification,such as ob⁃serving the morphology of DC under optical microscope,analyzing their surface markers by flow cytometry,detecting the secretion of cyto⁃kines with ELISA kit,and determining the ability to stimulate lymphocyte proliferation with the CCK-8 method.The results showed that the DC with high phagocytic activity could be induced under the condition of 20 ng/mL IL-4 and 40 ng/mL GM-CSF.The rhomboid cells with surface spike could be seen after 3 days of cultivation inducing.Mature DC with a typical dendritic structure was obtained after LPS stimula⁃tion;meanwhile,the expression levels of surface marker molecules CD11c,CD80 and MHCⅡwere achieved by 80.08%,81.27%and 91.47%,respectively.Additionally,the IL-2 and IFN-γthat secreted in the culture system increased significantly(P<0.05).Finally,the CCK-8 assay results showed that mature DC were mixed with lymphocytes at a ratio of 1:5,which could significantly stimulate cell prolifer⁃ation.To sum up,the inducing cultivation method of duck bone marrow-derived DC was successfully established in vitro in this study,and the prepared cells possessed the basic biological functions of dendritic cells.