“自体血清+鸡尾酒法”诱导外周血DC成熟的研究

Experimentary Study of Isolating Dendritic Cells from Peripheral Blood and Inducing Them to Mature by the Way Named "Autologous Serum+Cocktail"in Vitro

目的比较三种不同的分离诱导外周血树突状细胞(DC)成熟的方法。方法取正常成人外周血约180ml,分成三组。分离单核细胞后,经典脂多糖(LPS)诱导组(A组):接种于含RPMI1640+胎牛血清完全培养基的25cm2培养瓶中,2h后弃悬浮细胞,将贴壁单核细胞,用粒-巨噬细胞集落刺激因子和白介素4培养,培养到第6天时加入LPS诱导24h;鸡尾酒诱导组(B组):单核细胞的培养同A组,培养到第6天时用细胞因子组合(1000U/ml肿瘤坏死因子、10ng/ml白介素6、10ng/ml白介素1β、1μg/ml前列腺素E2)诱导24h;自体血清+鸡尾酒诱导组(C组):单核细胞用RPMI1640+10%自体血清完全培养基培养,诱导方法同B组。用流式细胞仪检测各组收集的DC细胞成熟表型CD80、CD86、CD83以及白细胞表面抗原HLA-DR的表达;动态观察各组细胞成熟过程中形态的改变以及其激活淋巴细胞增殖的能力。结果C组DC成熟过程中形态好、突起多;其成熟表型CD86、CD83、CD80以及HLA-DR的平均表达率分别是(88.60±6.34)%、(50.55±4.03)%、(73.89±6.15)%、(93.24±7.78)%,明显高于A、B组(P<0.05);激活淋巴细胞增殖指数为2.03,亦明显高于A、B组(P<0.05)。结论自体血清+鸡尾酒诱导法是一种简单、方便、经济的外周血DC分离诱导成熟的新方法。

Objective To explore a good and effective method to isolate dendritic cells （DCs） from the peripheral blood and induce them to mature. Methods The peripheral blood （about 180ml） obtained from healthy donors （from 20 years to 40 years old） was divided into three groups. Peripheral blood mononuclear cells （PBMCs） were isolated.①Lipopolysaccharid （LPS） inducing groups（group A）： PBMCs were inoculated in 25cm^2 cell culture flask containing RPMI1640 and fetal calf serum（FCS）medium. Two hours later, the suspended cells were given up, the cells adhering to the wall were cultured in the medium containing granulocyte-macrophoge colony stimulating factor （GM-CSOF） and IL-4 for 6 days, and then they were induced by LPS for 24 hours in order to make them mature. ②Cocktail inducing group （group B）： culturing PBMCs are the same as group A. DCs were induced by cytokine mixture （1000 U/ml TNF-α, 10 ng/ml IL-6, 10 ng/ml IL-β, 1μg/ml PGE2） for 24 hours in order to make them mature.③autologous serum ＋ cocktail inducing group （group C）： PBMCs were cultured in the medium containing RPMI1640 and autologous serum （AS）, and then they were induced by the same way as the group B. The expressions of CD80, CD86, CD83 and HLA-DR in DCs collected from all the groups were determined respectively by flow cytometry. The change in shapes of DCs during the growth and their capability of activating lymphocytes to proliferate were dynamically observed. Results DCs in groups C had good shapes and many dendrites. The rates of the expression of CD80, CD86, CD83 and HLA-DR in group C were 88.60%, 50.55%, 73.89%, 93.24% respectively and they were significantly higher than those in the other two groups （P〈0.05）. The proliferation index of the activated lymphocytes in group C was 2.03, which was also significantly higher than those in the other two groups （P〈0.05）. Conclusion The inducement by using autologous serum ＋ cocktail is a new and good method to induce DCs, which are isolated from peripheral blood, to mature.