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目的:探讨IL-27对人外周血单个核细胞来源的树突状细胞(dendritic cell,DC)形态和功能的影响及其作用机制。方法:从正常健康人外周血中分离出单个核细胞,将其用GM-CSF、IL-4体外培养7 d,并于培养的第5天加入不同刺激因子,并将细胞分为4组:阴性对照组、阳性对照组(20 ng/ml TNF-α)、IL-27(20 ng/ml)组、IL-27+TNF-α组(即双细胞因子组,10ng/ml TNF-α+10 ng/ml IL-27)。用倒置显微镜观察培养7 d的DC形态,用流式细胞术检测DC表面共刺激分子CD1a/CD83和CD80/CD86的水平,RT-PCR检测DC表面趋化因子受体CCR5、CCR7 mRNA的表达,混合淋巴细胞实验检测DC刺激同种异体T淋巴细胞增殖的能力,Western blotting检测DC信号通路蛋白P-STAT1/STAT3的含量。结果:IL-27组和双细胞因子组诱导7 d时DC呈现典型的成熟形态学特征;DC表面CD1a和CD83双阳性表达[(35.75±4.10)%、(52.49±2.65)%vs(23.29±4.49)%,P<0.05]、CD80和CD86双阳性表达[(39.06±1.61)%、(54.10±0.46)%vs(22.66±3.20)%,P<0.05]、趋化因子受体CCR7 mRNA[3.98±0.09、4.75±0.11 vs 3.09±0.18,P<0.05]和转录因子蛋白P-STAT1/STAT3水平均较阴性对照组明显上调,而CCR5 mRNA[0.99±0.03、0.61±0.02 vs 1.23±0.26,P<0.05]表达含量则明显下降;IL-27组和双细胞因子组DC均可明显刺激T细胞增殖,且随DC与T细胞比例增加而增强,以双细胞因子组的刺激作用更为明显。结论:细胞因子IL-27可以直接或者协同TNF-α诱导人DC分化成熟,并增强DCs的抗原提呈功能,其机制可能与活化P-STAT1/STAT3信号通路有关。
Objective: To investigate the effect of IL-27 on the morphology and function of dendritic cells (DCs) derived from human peripheral blood mononuclear cells and its mechanism. Methods: Mononuclear cells were isolated from normal healthy human peripheral blood and cultured with GM-CSF and IL-4 for 7 days in vitro. Different stimulating factors were added on the 5th day of culture. The cells were divided into 4 groups: Negative control group, positive control group (20 ng / ml TNF-α), IL-27 (20 ng / ml) 10 ng / ml IL-27). The morphology of DCs cultured on day 7 was observed with inverted microscope. The levels of CD1a / CD83 and CD80 / CD86 on DCs were detected by flow cytometry. The expressions of CCR5 and CCR7 mRNA on DCs were detected by RT-PCR. The ability of DC to stimulate allogeneic T lymphocyte proliferation was tested by mixed lymphocyte assay. The content of P-STAT1 / STAT3 in DCs was detected by Western blotting. Results: DCs showed typical mature morphological features at 7 days after induced by IL-27 and double-cytokines. Double positive expression of CD1a and CD83 on DCs [(35.75 ± 4.10)% vs (52.49 ± 2.65)% vs (23.29 ± (49.06 ± 1.61)%, (54.10 ± 0.46)% vs (22.66 ± 3.20)%, P <0.05]. The chemokine receptor CCR7 mRNA [ 3.98 ± 0.09, 4.75 ± 0.11 vs 3.09 ± 0.18, P <0.05], and the level of P-STAT1 / STAT3 protein were significantly up-regulated compared with the negative control group, while CCR5 mRNA [0.99 ± 0.03,0.61 ± 0.02 vs 1.23 ± 0.26, P <0.05], while the level of T-cell proliferation was significantly increased in both IL-27 and DC-2 groups, and increased with the increase of the ratio of DC to T cells. The stimulation with double cytokines was more obvious . CONCLUSION: The cytokine IL-27 can directly or synergize with TNF-α to induce human DC differentiation and maturation and enhance the antigen presenting function of DCs, which may be related to the activation of P-STAT1 / STAT3 signaling pathway.