骨肉瘤细胞外泌体激活肺组织成纤维细胞炎性信号通路

Osteosarcoma Cells Derived Exosome Activate Inflammatory Signaling Pathways in Lung Fibroblast Cells

【目的】本研究旨在研究骨肉瘤细胞来源外泌体对靶器官微环境的调控作用及其分子机制。【方法】提取骨肉瘤细胞143B外泌体并鉴定,通过PKH26荧光染色后经尾静脉注入裸鼠体内,24 h后提取肝、脾、肺、肾及脑组织,荧光显微镜下计数不同视野红色荧光数量;通过免疫荧光检测不同类型细胞中外泌体摄取情况;143B外泌体与人肺成纤维细胞共培养,荧光显微镜检测摄取情况,进一步利用qPCR检测炎性因子IL-1β、IL-6及TNF-α表达水平,通过蛋白印迹法检测炎性信号通路NF-κB通路p-p65和MAPK通路p-ERK及p-p38的变化。【结果】143B细胞外泌体中TSG101、Flotillin-1、CD63、CD9表达,Calnexin表达缺失(P<0.05);电镜外泌体呈“茶托”样形状,大小为(141.92±52.85)nm;外泌体经裸鼠尾静脉注射后进入肺组织含量显著高于肝、肾、脾、脑等器官(P<0.05);外泌体与人肺成纤维细胞共培养后炎性因子IL-1β、IL-6及TNF-αmRNA水平明显升高(P<0.05),同时p-p65,p-ERK及pp38均显著上调(P<0.05)。【结论】骨肉瘤细胞外泌体可激活肺成纤维细胞NF-κB及MAPK炎性信号通路,进而促进炎性因子IL-1β、IL-6及TNF-α产生。

【Objective】To investigate the function and molecular mechanism of osteosarcoma cells derived exosome on microenvironment of target organs.【Methods】The osteosarcoma derived exosomes were extracted and injected into nude mice through tail vein after PKH26 fluorescence staining.The liver,spleen,lung,kidney and brain tissues were extracted 24 hours later and then the amount of red fluorescence in different fields was counted under fluorescence microscope.The uptake of exosomes in different types of cells was detected by immunofluorescence.143B derived exosomes were co-cul⁃tured with human lung fibroblasts,and the uptake was detected by fluorescence microscopy.The expression levels of in⁃flammatory cytokines IL-1β,IL-6 and TNF-αwere detected by RT-qPCR,while the changes of p-p65 in inflammatory signaling pathway of NF-κB and p-ERK,p-p38 in MAPK signaling were detected by western blotting.【Results】TSG101,Flotillin-1,CD63 and CD9 were expressed in 143B derived exosomes,and Calnexin expression was absent(P<0.05).The exosomes presented a saucer-like structure under electron microscope.The size of the exosomes is(141.92±52.85)nm.The exosomes distributed more in lung tissue than liver,kidney,spleen and brain after injection through the tail vein of nude mice(P<0.05).The mRNA levels of inflammatory cytokines IL-1β,IL-6and TNF-αwere significantly increased in human lung fibroblast cells after incubation with 143B exosomes(P<0.05).p-p65,p-ERK and p-p38MAPK were significantly up-regulated(P<0.05).【Conclusions】Osteosarcoma cells derived exosomes could activate inflammatory signaling pathway NF-κB and MAPK,and up-regulate the expression of the inflammatory cytokines IL-1β,IL-6 and TNF-αin lung fibroblast cells.