热刺激后小胶质细胞细胞外囊泡对神经元的损伤作用  

Effect of microglial derived extracellular vesicles on neuronal damage after heat stress

作　　者：李萍[1] 罗雪[1] 罗珍 何根林[1] 王泽泽 申婷婷 刘晓倩 谭雨龙[1] 杨学森[1] LI Ping;LUO Xue;LUO Zhen;HE Genlin;WANG Zeze;SHEN Tingting;LIU Xiaoqian;TAN Yulong;YANG Xuesen(Department of Tropical Medicine,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)军事预防医学系热带医学教研室,重庆400038

出　　处：《陆军军医大学学报》2024年第18期2029-2035,共7页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(cstc2020jcyj-msxmX1020)。

摘　　要：目的探究热刺激后小胶质细胞细胞外囊泡对神经元损伤的影响。方法热刺激BV2小胶质细胞后收集上清,通过不同的超速离心速度获取细胞外大囊泡和小囊泡。利用纳米粒度分析仪检测粒径,利用Western blot检测囊泡上TSG101、CD63和flotillin-1的表达。PKH67标记BV2来源的囊泡后与N2a细胞共孵育,检测神经元对小胶质细胞细胞外囊泡的摄取情况。分别将热刺激后BV2来源的大囊泡和小囊泡与N2a共孵育,通过CCK-8、细胞外乳酸脱氢酶(lactate dehydrogenase,LDH)、台盼蓝染色和TUNEL染色法评价热刺激后N2a的损伤情况。结果小囊泡粒径介于30~120 nm,高表达TSG101和CD63,大囊泡粒径介于90~1000 nm,高表达flotillin-1;BV2来源的细胞外囊泡可被N2a细胞摄取并参与热刺激对N2a细胞损伤的调节,其中CCK-8检测结果表明小胶质细胞来源的大小囊泡均能降低热刺激后N2a细胞的活力(P<0.05)。LDH检测、台盼蓝染色及TUNEL检测结果表明大囊泡(P<0.05)和小囊泡(P<0.01)均能显著提高热刺激后N2a细胞LDH的释放水平、N2a细胞的蓝染水平及凋亡程度,且小囊泡处理组中N2a细胞的LDH释放水平、蓝染和凋亡水平高于大囊泡处理组。结论热刺激后小胶质细胞通过细胞外囊泡加剧了神经元的损伤。ObjectiveTo investigate the effect of microglial derived extracellular vesicles on neuronal damage in the context of heat stress.MethodsAfter BV2 microglial cells were exposed to heat stress,the supernatant was collected and subjected to ultracentrifugation at different speeds to obtain large and small vesicles,respectively.Nano Particle Tracking and Zeta Potential Distribution Analyzer was used to measure and analyze the size distribution of the large vesicles and small vesicles.Western blotting was used to detect the expression of specific vesicle surface markers,TSG101,CD63 and flotillin-1.Microglial extracellular vesicles were labeled with PKH67 dye and then co-cultured with N2a cells to examine the uptake by capacity the neurons.After large and small vesicles derived from microglia after heat stress stimulation were co-cultured with N2a cells,respectively,CCK-8 assay,lactate dehydrogenase(LDH)assay,Trypan blue staining and TUNEL assay were employed to evaluate heat stress induced neuronal damage.ResultsThe small vesicles were in a particle size of 30~120 nm,and highly expressed TSG101 and CD63,whereas the large vesicles,in a size of 90~1000 nm,highly expressed flotillin-1.The BV2-derived extracellular vesicles could be taken up by N2a cells and were proved to be involved in the modulation of N2a cell injury caused by heat stress.CCK-8 assay showed that both large and small vesicles of microglial cells inhibited the viability of N2a cells after heat exposure(P<0.05).The results of LDH assay,Trypan blue staining and TUNEL assay showed that both large(P<0.05)and small vesicles(P<0.01)significantly enhanced the LDH release,blue stain intensity and apoptosis of N2a cells after heat exposure,and the release,intensity and apoptosis were stronger in the cells treated with small vesicles than those group of large vesicles.ConclusionMicroglia aggravate heat stress-induced neuronal damage through releasing extracellular vesicles.

关 键 词：小胶质细胞 细胞外囊泡 热刺激 神经元损伤 

分 类 号：R338.1[医药卫生—人体生理学] R339.52[医药卫生—基础医学] R363.21