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目的:通过体外培养肿瘤患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)来源的DC,探讨VEGF-C/VEGFR3信号通路对DC功能的影响。方法:采用GM-CSF和IL-4共同诱导的方法培养DC,分别采用VEGF-C(VEGF-C-DC),LPS(LPS-DC)或LPS+VEGF-C(LPS+VEGF-C-DC)诱导,同时设未处理组即未成熟DC细胞(imDC);流式细胞术检测DC表面CD80、CD83、VEGFR3和TLR4的表达情况,同时采用免疫荧光法检测VEGFR3在DC的表达;ELISA方法检测不同处理组的DC上清中IL-6、TNF-α、IL-12的分泌情况。结果:LPS-DC高表达VEGFR3;LPS-DC和LPS+VEGF-C-DC高表达CD80和CD83,明显高于imDC(18.56%vs 8.52%,P<0.05),显示出成熟DC的表型特征;与LPS-DC相比,LPS+VEGFC-DC表面TLR4的表达下调,LPS+VEGF-C-DC上清液中细胞因子IL-6、TNF-α和IL-12的分泌减少。结论:VEGF-C/VEGFR3通路通过降低DC表面TLR4的表达减少其细胞因子的分泌,对DC起负向免疫调控作用。
OBJECTIVE: To investigate the effect of VEGF-C / VEGFR3 signaling pathway on DC function in vitro by culturing DCs derived from peripheral blood mononuclear cells (PBMCs) from patients with cancer. Methods: DCs were induced by co-administration of GM-CSF and IL-4. The cells were treated with VEGF-C, LPS-DC or LPS + VEGF-C (ImDC). Flow cytometry was used to detect the expression of CD80, CD83, VEGFR3 and TLR4 on DCs. The expression of VEGFR3 in DCs was detected by immunofluorescence method. The secretion of IL-6, TNF-α and IL-12 in the DC supernatants of different treatment groups. Results: LPS-DCs highly expressed VEGFR3; CD80 and CD83 were highly expressed in LPS-DCs and LPS + VEGF-C-DCs, which were significantly higher than those in imDC (18.56% vs 8.52%, P <0.05) Compared with LPS-DC, the expression of TLR4 on LPS + VEGFC-DC was down-regulated, while the secretion of IL-6, TNF-α and IL-12 in LPS + VEGF-C-DC supernatant was decreased. CONCLUSION: The VEGF-C / VEGFR3 pathway can reduce the secretion of cytokines by decreasing the expression of TLR4 on DCs, and negatively regulate DCs.