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目的探讨并比较不同细胞因子组合、诱导时间对树突状细胞(DC)体外诱导、成熟,分泌细胞因子水平和刺激淋巴细胞增殖能力的影响,为建立DC体外高效、规模化培育体系提供依据。方法分离健康人外周血单个核细胞(PBMC),贴壁法获得DC前体细胞,用白细胞介素4(IL-4)和粒细胞巨噬细胞集落刺激因子(GM-CSF)诱导2 d,将其分为6组。A组加入肿瘤坏死因子α(TNF-α)、IL-1α和前列腺素E2(PGE2);B组加入α干扰素(IFN-α);C组加入脂多糖(LPS);3组细胞继续培育2 d。D~F组细胞先进行1/3体积换液,24 h后,分别再加入上述A~C组的成熟因子,再继续诱导2 d。分别收获各组上清和细胞,进行细胞计数,采用流式细胞术进行表型分析;采用ELISA检测各组DC分泌IL-12p70的水平、采用细胞增殖检测试剂盒刺激淋巴细胞增殖的能力。结果 6组体外诱导体系均能培育出形态学类似DC的细胞,DC数量与纯度(均达到96%以上),各组间无显著性差异。A组与D组比较:CD40、CD80、CD83、CD86共刺激分子表达水平相近,但D组DC分泌IL-12p70水平高于A组;B组与E组比较:E组DC的CD86表达水平显著高于B组,CD80表达量略高于B组,2组DC分泌IL-12p70的水平相近;C组与F组比较:CD40、CD80、CD83、CD86共刺激分子表达水平和分泌IL-12p70的水平均相近。6组DC刺激淋巴细胞增殖水平均相近。三种不同成熟因子(组合)比较:B组DC共刺激分子CD80水平明显低于C组和A组,且IL-12p70分泌水平最低,而C组和A组共刺激分子表达水平与IL-12p70分泌水平相近。结论 TNF-α、IL-1α和PGE-2三种细胞因子联合以及LPS可有效地诱导DC表达高水平免疫共刺激分子、分泌高水平IL-12p70。
Objective To explore and compare the effects of different combinations of cytokines and inducing time on the induction, maturation and cytokine secretion of dendritic cells (DCs) in vitro and to stimulate the proliferation of lymphocytes, and to provide a basis for establishing an efficient and scaled cultivation system of DC in vitro. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood mononuclear cells (PBMCs). DC precursor cells were obtained by adherent method. The cells were induced with interleukin-4 and GM-CSF for 2 days, Divide it into 6 groups. In group A, tumor necrosis factor α (TNF-α), IL-1α and prostaglandin E2 (PGE2) were added; in group B IFN-α was added; in group C, lipopolysaccharide (LPS) was added; 2 d. Cells in group D ~ F were subjected to 1/3 volume exchange first. After 24 h, maturation factors of group A ~ C were added respectively and then induced for another 2 days. The supernatants and cells of each group were harvested for cell counting, and the phenotypes were analyzed by flow cytometry. The levels of IL-12p70 secreted by DC in each group were detected by ELISA, and the ability of lymphocyte proliferation was stimulated by cell proliferation assay kit. Results All of the 6 in vitro induction systems were able to cultivate cells with morphologically similar DCs. The number and purity of DCs (both above 96%) were not significantly different among groups. The expression of CD40, CD80, CD83 and CD86 costimulatory molecules were similar in group A and group D, but the level of IL-12p70 in group D was higher than that in group A. The expression of CD86 in group E was significantly higher than that in group E The expression level of CD80 in group C was slightly higher than that in group B, and the levels of IL-12p70 secreted by two groups were similar. The expression of costimulatory molecules CD40, CD80, CD83, CD86 and IL-12p70 The levels are similar. 6 groups of DC stimulation of lymphocyte proliferation were similar. Three different maturation factors (combination): B group DC costimulatory molecule CD80 levels were significantly lower than the C group and A group, and IL-12p70 secretion was the lowest, while the C group and A group co-stimulatory molecules and IL-12p70 Secretion level is similar. Conclusion The combination of TNF-α, IL-1α and PGE-2 cytokines and LPS can effectively induce DCs to express high level of immune costimulatory molecules and secrete high levels of IL-12p70.