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探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。
To investigate the role of trichosanthin (Tk) combined with CD40 signaling in the induction of maturation and activation of human monocyte-derived DC (MoDC) and its role in mediating Th2 cell differentiation. Human MoDCs were induced in vitro using a combination of GM-CSF and IL-4, and DCs loaded with Tk were stimulated with mouse anti-human CD40 challenge mAb (5C11) to prepare mature DCs. The ability of DC phenotypes (CD80, CD83, CD86, CD14, PDL1) and their uptake of FITC-Dextran was analyzed by immunofluorescence labeling and flow cytometry; IL-12p70 content in DC supernatants was determined by ELISA; intracellular staining and The proportion of CD4 + IFN-γ + T and CD4 + IL-4 + T in mature DC activated T cells was detected by flow cytometry. The experimental results show that Tk alone can not stimulate complete maturation of DCs, and the use of Tk-loaded DCs must be combined with exogenous mature stimulation signals (eg, 5C11) to effectively promote DC maturation, which is manifested in the up-regulated expression of CD80, CD83, and CD86, and down-regulated expression of CD14. The ability of DC to take up FITC-Dextran is reduced. Compared with the CD40 signal alone group, the PDL1 molecule on the mature DC surface induced by Tk combined with CD40 signal was down-regulated, the ability to secrete IL-12 was significantly reduced, and the proportion of CD4+IL-4+T in T cells was significantly increased. This shows that mature MoDC loaded with Tk can effectively mediate Th2 cell differentiation in vitro.