VEGF-C/VEGFR3通路对肿瘤患者外周血来源DC的影响

Influence of VEGF-C/VEGFR3 signaling pathway on dendritic cells derived from peripheral blood of tumor patients

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 culture peripheral blood mononuclear cell（ PBMC） derived DCs of cancer patients in vitro and investigate the effect of VEGF-C / VEGFR3 signaling pathway on the functions of DCs. Methods： DCs were cultured using GM-CSF and IL-4 co-stimulating method,and then induced by VEGF-C（ VEGF-C-DC）,LPS（ LPS-DC） or LPS ＋VEGF-C（ LPS ＋ VEGF-C-DC）,respectively; and immature DCs（ im DCs） was used as control group. The expressions of CD80,CD83,VEGFR3 and TLR4 on DCs surface were evaluated by flow cytometry. The expression of VEGFR3 was detected by immunofluorescence method. The secretions of IL-6,TNF-α and IL-12 in different treatment groups were detected by ELISA method. Results： VEGFR3 was highly expressed in LPS-DC. The expressions of CD80 and CD83 on LPSDC and LPS ＋ VEGF-C-DC were much higher than that on im DC and VEGF-C-DC（ 18. 56 % vs 8. 52 %,P〈0. 05）,and showed phenotypic characteristics of mature DCs. Moreover,compared with LPS-DC,the expression of TLR4 on LPS＋ VEGF-C-DC was down-regulated,and the secretion of IL-6,TNF-α and IL-12 in LPS ＋ VEGF-C-DC supernatant were also decreased. Conclusion： The VEGF-C / VEGFR3 signaling pathway can decrease DCs cytokine secretion through down-regulating TLR4 expression and act as a negative regulator of DCs immune response.