热损伤血管内皮细胞对巨噬细胞极化的影响

The effects of vascular epithelial cell after thermal injury on the polarization of macrophage

目的探讨血管内皮细胞热损伤后培养上清对巨噬细胞极化的影响及机制。方法小鼠肺微血管内皮细胞系(MPVECs)给予40℃热刺激不同时间后,观察细胞状态和活力,ELISA检测培养上清中趋化因子5(CCL5)含量,选取合适的刺激时间;构建MPVECs热处理上清与小鼠巨噬细胞系(RAW264.7)共培养体系,对照组予以正常培养3 h上清,处理组予以40℃处理3 h上清,干预组予以40℃处理3 h上清,同时加入Maraviroc,阻断CCL5/趋化因子5受体(CCR5)轴,培养6 h后流式细胞术观察巨噬细胞M1/M2极化,qPCR检测促炎相关细胞因子mRNA表达,ELISA检测培养上清细胞因子含量。结果MPVECs给予40℃热处理3 h细胞形态较完整,培养上清CCL5含量较高,适合用于刺激RAW264.7细胞;处理组、干预组予以不同处理后RAW264.7细胞M1极化明显上升(P<0.01),予以Maraviroc,阻断CCL5/CCR5轴,M1极化下降(P<0.01)。处理组、干预组予以不同处理后促炎细胞因子IL-1β、IL-6、TNF-α表达和培养上清含量均上升(P<0.05),阻断CCL5/CCR5途径明显减少IL-1β的表达(P<0.01)。结论血管内皮细胞受到热损伤时可以释放CCL5,通过CCL5/CCR5轴调控巨噬细胞向促炎的M1型极化并释放促炎细胞因子,靶向阻断CCL5/CCR5轴抑制巨噬细胞M1极化和相关促炎细胞因子分泌。

Objective To investigate the effects of vascular epithelial cell-conditioned medium after thermal injury on the polarization of macrophage.Methods Mouse pulmonary microvascular endothelial cell lines(MPVECs)were subjected to heat stimulation at 40℃for different periods of time,and the cell status and viability were observed.The content of chemokine ligand 5(CCL5)in the cultural supernatant was detected by ELISA.The co-culture system of the MPVECs conditioned medium after heat treatment with mouse macrophage cell line(RAW264.7)was constructed.The control group had normally cultured(3 h)conditioned medium,the treatment group contained conditioned medium treated with 40℃for 3 h,and the intervention group was the conditioned medium(40℃for 3 h)combined with Maraviroc administration to hinder the CCL5/CCR5 axis.Following 6 h culturing,flow cytometry was performed to check macrophage M1/M2 polarization,qPCR detected the mRNA expression of pro-inflammatory cytokines,and ELISA assessed the cytokine content of cultural supernatant.Results MPVECs treated with heat at 40℃for 3 h had a relatively complete cell morphology,combined with a higher CCL5 content in the cultural supernatant,which were suitable for RAW264.7 cell stimulation.The M1 polarization of RAW264.7 cells increased significantly in the treatment group and the intervention group(P<0.01),which were further declined by Maraviroc administration in the intervention group(P<0.01).Moreover,the expression of pro-inflammatory cytokines IL-1β,IL-6,TNF-αin the RAW264.7 cells and the conditioned medium after co-culturing was all increased in the treatment group and the intervention group(P<0.05),and further blocking of CCL5/CCR5 pathway significantly reduced IL-1βexpression(P<0.01).Conclusions Vascular endothelial cells release CCL5 after thermal injury,which regulates the macrophage to pro-inflammatory M1 polarization and releases pro-inflammatory cytokines through CCL5/CCR5 axis,Targeted blocking of the CCL5/CCR5 axis inhibits the M1 polarization of macrophages and the release of related pro-inflammatory cytokines.