HMGB1激活肿瘤相关巨噬细胞表面RAGE促进淋巴管生成

HMGB1 promotes lymph angiogenesis through the activation of RAGE on tumor-associated macrophages

目的:探讨高迁移率族蛋白B1(high mobility group box-1,HMGB1)与肿瘤相关巨噬细胞(tumorassociated macrophage,TAMs)促进人淋巴管内皮细胞(human lymphatic endothelial cell,HDLEC)增殖、迁移及淋巴管生成的关系以及作用机制。方法:沉默人单核细胞株(THP-1)的晚期糖基化终产物受体(receptor for advanced glycation end,RAGE)基因,诱导RAGE^+/−THP-1细胞分化为RAGE^+/−M0巨噬细胞和RAGE^+/−TAMs;用RT-PCR检测IL-10,IL-12,IL-1β,CCL-13表达、ELISA检测TGF-β,IL-23,IL-10,TNF-α分泌量;蛋白质印迹检测RAGE,CD163,CD206蛋白水平。6种条件培养基及对照组培养HDLEC,CCK-8实验、细胞迁移实验、体外侵袭实验观察HDLEC增殖能力、迁移能力及成管能力的变化。结果:与M0巨噬细胞相比,TAMs高表达IL-10及CCL-13 mRNA及低表达IL-12及IL-1βmRNA;上清液中TGF-β及IL-10 mRNA也增加(P<0.05)。与对照组相比,由HMGB1刺激的RAGE+TAMs上清液孵育的HDLEC增殖及迁移能力最强,形成淋巴管数目最多(P<0.05);HMGB1刺激的RAGE−TAMs上清液促进HDLEC增殖及迁移能力明显比HMGB1刺激的RAGE+TAMs上清液促进HDLEC增殖及迁移能力更弱(P<0.05);RAGE−TAMs上清液能明显减少HMGB1介导的促HDLEC形成的淋巴管数目(P<0.05)。与对照组相比,HMGB1刺激的RAGE+TAMs上清液中VEGF-C的含量最高(P<0.05)。结论:HMGB1刺激TAMs表面的RAGE促进HDLEC增殖、迁移及淋巴管形成,该过程中细胞因子VEGF-C起重要作用。

Objective:To evaluate whether the high mobility group box-1(HMGB1)and tumor-associated macrophages(TAMs)involved in proliferation,migration and lymph angiogenesis of HDLEC and its mechanism.Methods:THP-1 cells expressed receptor for advanced glycation end(RAGE)gene was knocked down and then polarized to M0 macrophages and TAMs.The mRNA expression of IL-10,IL-12,IL-1βand CCL-13 by RT-PCR.The protein levels of IL-23,IL-10,TGF-β,TNF-αin supernatant of M0 macrophages and TAMs by ELISA.CD163,CD206 and RAGE protein expression in TAMs and M0 macrophages by Western blot.Six conditioned media were collected for human dermal lymphatic endothelial cells(HDLEC)proliferation,migration,matrigel assay and VEGF concentration analysis.Results:Compared with M0 macrophages,TAMs are overexpressed IL-10,CCL-13 mRNA and low expression of IL-12 and IL-1βmRNA.The secretion of TGF-βmRNA and IL-10 mRNA increased in the supernatant(P<0.05).Conditioned medium from HMGB1-stimulated RAGE+TAMs activated lymph angiogenesis by upregulating the VEGF compared to M0 macrophages(P<0.05).On the contrary,RAGE knockout obviously decreased the corresponding effects of HMGB1 preconditioned TAMs upon HDLEC.Conclusion:HMGB1 promotes the proliferation,migration and lymphatic formation of HDLEC by stimulating the RAGE receptor on TAMs and VEGF-C may be involved in that pathway.