LM-Exos对巨噬细胞和CD8^(+)T淋巴细胞的调节作用  

Regulatory roles of LM-Exos on macrophages and CD8^(+)T lymphocytes

作　　者：焦健 李能秀 左雨霏 马忠梅 常意行 张石磊 孟庆玲[1] 才学鹏[2] 乔军[1] JIAO Jian;LI Neng-xiu;ZUO Yu-fei;MA Zhong-mei;CHANG Yi-hang;ZHANG Shi-lei;MENG Qing-ling;CAI Xue-peng;QIAO Jun(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832003,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国兽医药品监察所,北京100081

出　　处：《南方农业学报》2024年第12期3738-3747,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32160819)。

摘　　要：【目的】分离感染单核细胞增多性李斯特菌(Listeria monocytogenes,LM)RAW264.7巨噬细胞分泌的外泌体(Exosomes,Exos),探究其对RAW264.7巨噬细胞和CD8^(+)T淋巴细胞的调节作用及机制,为揭示LM感染、胞内生存及免疫逃逸机制提供理论参考。【方法】通过差异超速离心法结合分子排阻法提取感染LM后RAW264.7巨噬细胞外泌体(LM-Exos),利用透射电子显微镜、粒径分析仪和Western blotting鉴定LM-Exo表型。通过实时荧光定量PCR、细胞活力检测和ELISA等方法分析不同剂量LM-Exos对RAW264.7巨噬细胞和CD8^(+)T淋巴细胞的调节作用。【结果】LM-Exos具有双层膜结构类圆形的小囊泡,直径约为60~80 nm。Western blotting检测结果显示,LM-Exos表面含有CD9、CD63、TSG101特异性标记蛋白。LM-Exos对RAW264.7巨噬细胞形态和功能的影响具有明显的浓度依赖性,随着LM-Exos刺激时间延长,细胞形态由圆形逐渐分化为长梭形,并伸出不同形态的伪足。实时荧光定量PCR结果显示,随着LM-Exos刺激浓度的增加,IL-1β、IL-10、IL-6和TNF-α基因相对表达量均呈升高趋势。高浓度LM-Exos刺激后,RAW264.7巨噬细胞活力略有下降但抗LM EGD-e能力增强。与低浓度LM-Exos刺激相比,高浓度LM-Exos刺激显著(P<0.05)或极显著(P<0.01)提高了CD8^(+)T淋巴细胞TNF-α和IL-10相对分泌水平,并诱导了CD8^(+)T细胞形态变化及凋亡水平升高。【结论】不同剂量的LM-Exos对RAW264.7巨噬细胞和CD8^(+)T淋巴细胞表现出不同的调控作用。LM-Exos能增强RAW264.7巨噬细胞抗LM EGD-e能力,同时也能诱导CD8^(+)T淋巴细胞分化和凋亡。LM-Exos可以作为免疫激活剂,增强RAW264.7巨噬细胞的免疫功能,高剂量的LM-Exos可能会通过诱导T细胞凋亡来减弱机体的免疫应答,进而实现LM免疫逃逸,这种双重作用可能是LM在感染过程中实现免疫逃逸的重要机制。【Objective】To isolate exosomes(Exos)secreted by RAW264.7 macrophages infected with Listeria monocytogenes(LM),and to explore their regulatory effects and mechanisms on RAW264.7 macrophages and CD8^(+)T lymphocytes,which could provide theoretical reference for revealing the mechanisms of LM infection,intracellular survival and immune escape.【Method】The exosomes of RAW264.7 macrophages infected with LM(LM-Exos)were extracted by differential ultracentrifugation combined with molecular exclusion method,and the phenotype of LM-Exos was identified by transmission electron microscopy,particle size analyzer and Western blotting.The regulatory effects of different doses of LM-Exos on RAW264.7 macrophages and CD8^(+)T lymphocytes were analyzed by real-time fluorescence quantitative PCR,cell viability detection and ELISA.【Result】LM-Exos had a double-layered membrane structure,a round-like vesicle with a diameter of approximately 60-80 nm.Western blotting results showed that the surface of LM-Exos contained CD9,CD63 and TSG101 specific marker proteins.The effect of LM-Exos on the morphology and function of RAW264.7 macrophages was obviously concentration-dependent.As the LM-Exos stimulation time increased,the cell morphology gradually differentiated from round to long spindle-shaped,and showed pseudopodia of different shapes.Real-time fluorescence quantitative PCR results showed that with the increase of LM-Exos stimulation concentration,the relative expression of IL-1β,IL-10,IL-6 and TNF-αgenes showed increasing trend.Stimulated by high-concentration LM-Exos,the viability of RAW264.7 macrophages slightly decreased but their resistance to LM EGD-e increased.Compared with the low-concentration LM-Exos stimulation group,high-concentration LM-Exos stimulation significantly(P<0.05)or extremely significantly(P<0.01)increased the relative secretion levels of TNF-αand IL-10 in CD8^(+)T lymphocytes,and induced morphological changes in CD8^(+)T lymphocytes,and increased levels of apoptosis.【Conclusion】Different dos

关 键 词：单核细胞增多性李斯特菌 外泌体 巨噬细胞 CD8^(+)T淋巴细胞 

分 类 号：S865.13[农业科学—野生动物驯养]