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目的研究外源性白三烯B4(LTB4)刺激条件下对小鼠体外诱导培养的树突状细胞(DCs)趋化功能的作用及肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)两种细胞因子表达及分泌量的影响,利用其受体BLT2通路探究其作用机制。方法体外原代培养小鼠骨髓来源单个核细胞,添加粒-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素-4(IL-4)诱导为DCs,磷酸脂多糖(LPS)体外刺激其成熟,分析DCs表面因子的表达;利用小干扰RNA(siRNA)阻断LTB4-BLT2通路后RT-PCR分析BLT2基因水平抑制情况;外源性LTB4刺激阻断通路的细胞,Tran-swell趋化小室结合噻唑蓝(MTT)法分析对其趋化功能的影响;ELISA检测不同条件对细胞TNF-α、IL-1β分泌量的影响,RT-PCR分析其基因水平表达的影响。结果体外诱导并刺激成熟的DCs高表达MHC-Ⅱ、CD11c、CD86。利用DNA干扰(RNAi)技术能够成功阻断LTB4-BLT2通路。外源性LTB4分别作用于正常表达和阻断BLT2通路的细胞,可明显促进BLT2正常表达DCs的趋化(P<0.05),且与剂量成正比。阻断通路的细胞趋化作用明显被抑制甚至消失(P<0.05);阻断通路的DCs在外源性LTB4刺激下,细胞TNF-α、IL-1β两种因子分泌量较之对照组明显降低(P<0.05),同时,这两种细胞因子在基因水平上的表达也明显下调。结论 LTB4刺激的DCs,其BLT2通路明显介入其趋化,同时,DCs分泌细胞因子的功能也与LTB4-BLT2通路相关。抑制BLT2基因表达,DCs的趋化移行作用和致炎因子的分泌功能均受到抑制,深入了解其机制可能为研究炎症有关药物提供新靶点。
Objective To study the chemotactic function of dendritic cells (DCs) cultured in vitro induced by exogenous leukotriene B4 (LTB4) and the effect of tumor necrosis factor-α (TNF-α), interleukin- 1β (IL-1β) expression and secretion of two cytokines, using its receptor BLT2 pathway to explore its mechanism of action. Methods Primary cultured murine bone marrow-derived mononuclear cells were stimulated with GM-CSF and IL-4 to induce DCs and phospholipase LPS in vitro The expression of DCs was analyzed by flow cytometry. The LTB4-BLT2 pathway was blocked by small interfering RNA (siRNA), and the inhibition of BLT2 gene expression was analyzed by RT-PCR. The exogenous LTB4 stimulated the blocked cells, Tran-swell chemotaxis The effect of different conditions on the secretion of TNF-α and IL-1β by ELISA was analyzed by ELISA and the gene expression level by RT-PCR. Results In vitro induction and stimulation of mature DCs high expression of MHC-Ⅱ, CD11c, CD86. The LTB4-BLT2 pathway can be successfully blocked by using DNA interference (RNAi) technology. Exogenous LTB4 could promote the chemotaxis of BLT2 normal DCs (P <0.05), which was directly proportional to the dose, on the cells that normally expressed and blocked the BLT2 pathway. The chemotactic effect of blocking pathway was significantly inhibited or even disappeared (P <0.05). The secretion of TNF-α and IL-1β in DCs blocked by exogenous LTB4 was significantly lower than that of control (P <0.05). At the same time, the expression of these two cytokines at the gene level was also significantly down-regulated. Conclusions LTB4-stimulated DCs are significantly involved in chemotaxis of BLT2 pathway, meanwhile, cytokine secretion by DCs is also related to LTB4-BLT2 pathway. Inhibition of BLT2 gene expression, DCs chemotaxis and secretion of inflammatory cytokines were inhibited, in-depth understanding of the mechanism may provide a new target for the study of inflammatory drugs.