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目的观察人外周血单核细胞分化为树突状细胞(DC)过程中,核转录因子-κB p50(NF-κB p50)的表达以及柴胡皂苷a对单核细胞分化为树突状细胞的影响。方法非连续密度梯度离心获取人外周血单核细胞;用含粒单细胞刺激因子(GM-CSF)、IL-4、肿瘤坏死因子(TNF-α)和柴胡皂苷a的RPMI-1640培养液分别对细胞因子组、柴胡皂苷组、联合培养组的单核细胞进行体外培养;应用倒置相差显微镜、瑞氏染色和CD14、CD83、HLA-DR、S-100的免疫细胞化学法鉴定单核细胞和DC;动态观察单核细胞分化为DC过程中不同时间点的NF-κB p50的表达,并进行图像分析。结果CD14、CD83、HLA-DR、S-100免疫细胞化学法证实所获单核细胞和DC符合各自的形态和表型特征。在含GM-CSF和IL-4的RPMI-1640培养液培养7d时,单核细胞分化成未成熟DC(imDC);在含GM-CSF、IL-4和TNF-a的RPMI-1640培养液继续培养3d时,imDC分化为成熟DC(mDC),两者的细胞核内NF-κB p50表达具有显著性差异(P<0.05)。单纯用只含柴胡皂苷a的RPMI-1640培养液培养单核细胞至7d时,可见其细胞核呈NF-κBp50阳性,但未见其分化为DC。若用含GM-CSF、IL-4和柴胡皂苷a的RPMI-1640培养液联合培养7d时,则该单核细胞分化为imDC;加入TNF-α后继续培养3d,imDC的细胞核呈NF-κB p50强阳性,免疫细胞化学法证实imDC已经分化为mDC。联合培养组细胞的表型表达均强于只加细胞因子组(P<0.05)。结论人外周血单核细胞分化为imDC和mDC过程中,细胞核内NF-κB p50表达逐渐增强。单纯用低浓度柴胡皂苷a不能刺激单核细胞分化为DC,但联合细胞因子GM-CSF、IL-4和TNF-α培养时,可使其分化为imDC和mDC。
Objective To observe the expression of nuclear factor-κB p50 (NF-κB p50) and the differentiation of monocytes into dendritic cells in the process of differentiation of human peripheral blood mononuclear cells into dendritic cells (DCs). influences. Methods Human peripheral blood mononuclear cells were obtained by discontinuous density gradient centrifugation; RPMI-1640 medium containing GM-CSF, IL-4, tumor necrosis factor (TNF-α) and saikosaponin-a Mononuclear cells of cytokine group, saikosaponin group and co-cultivation group were cultured in vitro; Inverted phase contrast microscopy, Wright’s staining, and CD14, CD83, HLA-DR, S-100 immunocytochemistry were used to identify mononuclear cells. Cells and DCs; Dynamically observe the expression of NF-κB p50 at different time points during the differentiation of monocytes into DCs, and perform image analysis. Results CD14, CD83, HLA-DR, S-100 immunocytochemistry confirmed that the obtained monocytes and DCs conformed to their respective morphological and phenotypic characteristics. When cultured in RPMI-1640 medium containing GM-CSF and IL-4 for 7 days, monocytes were differentiated into immature DCs (imDC); in RPMI-1640 medium containing GM-CSF, IL-4 and TNF-a After 3 days of continuous culture, imDCs differentiated into mature DCs (mDCs). There was a significant difference in the expression of NF-κB p50 between the two nuclei (P<0.05). When cultured mononuclear cells were cultured in RPMI-1640 medium containing only saikosaponin-a for 7 days, the nucleus was positive for NF-κBp50, but no differentiation to DC was observed. If the RPMI-1640 medium containing GM-CSF, IL-4 and saikosaponin a was co-cultured for 7 days, the monocyte was differentiated into imDC; after adding TNF-α, cultured for 3 days, the nuclei of imDC were NF- κB p50 was strongly positive and immunocytochemistry confirmed that imDC had differentiated into mDC. The phenotypic expression of the cells in the co-cultivation group was stronger than that in the cytokine-only group (P<0.05). Conclusion During the process of differentiation of human peripheral blood mononuclear cells into imDC and mDC, the expression of NF-κB p50 in the nucleus gradually increased. The low concentration of saikosaponin a can not stimulate the differentiation of monocytes into DCs, but when combined with the cytokines GM-CSF, IL-4 and TNF-α, it can be differentiated into imDC and mDC.