低氧诱导间充质干细胞外泌体通过改变M1/M2巨噬细胞极化减少椎间盘细胞凋亡和衰老  

Hypoxic mesenchymal stem cell-derived exosomes decrease apoptosis and senescence by shifting M1/M2 macrophage polarization

作　　者：李放 姜帆 马新妞 杜哲 Li Fang;Jiang Fan;Ma Xinniu;Du Zhe(Trauma Center,Peking University People’s Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院创伤救治中心,北京100044

出　　处：《中华实验外科杂志》2024年第4期803-806,共4页Chinese Journal of Experimental Surgery

基　　金：北京大学人民医院研究与发展基金(RDJP2022-06、RDJ2022-31)。

摘　　要：目的观察低氧预处理是否可以提高间充质干细胞外泌体治疗椎间盘退变的效果,探讨外泌体和巨噬细胞极化的机制。方法建立外泌体、巨噬细胞、髓核细胞的非接触共培养系统,分为对照组、常氧组及低氧组,对照组为巨噬细胞+脂多糖(LPS)或巨噬细胞和髓核细胞共培养体系;常氧组为添加常氧外泌体的共培养体系;低氧组是添加低氧诱导外泌体的共培养体系。通过定量实时聚合酶链反应、流式细胞术、β-半乳糖苷酶染色、免疫荧光染色测定常氧外泌体/低氧诱导外泌体对巨噬细胞极化的影响,对髓核细胞凋亡及衰老的影响。组间计量数据比较采用t检验。结果低氧组CD86的表达水平显著低于常氧组(2.78±0.31比7.00±0.51,t=8.627,P<0.05),CD163表达水平显著高于常氧组(19.01±0.38比15.54±0.44,t=9.499,P<0.05)。流式细胞术显示,低氧组髓核细胞凋亡率显著低于常氧组(4.44±0.40比11.16±0.50,t=14.351,P<0.05)。β-半乳糖苷酶染色结果显示,低氧组髓核细胞衰老率显著低于常氧组(49.75±1.63比59.71±1.63,t=4.425,P<0.05)。免疫组织化学染色证实,低氧组可增加合成基因COL2(1.35±0.03比1.03±0.05,t=6.500,P<0.05)及蛋白聚糖(ACAN)的表达(1.66±0.05比1.03±0.05,t=13.141,P<0.05),降解基因基质金属蛋白酶(MMP)-13(0.80±0.04比1.00±0.05,t=5.496,P<0.05)及ADAMTS5的表达低于常氧组(0.84±0.02比1.00±0.05,t=6.723,P<0.05)。结论间充质干细胞外泌体在体外低氧条件下减少细胞凋亡和衰老,可能是通过M1/M2巨噬细胞的极化。Objective To observe whether hypoxic pretreatment can improve the efficacy of mesenchymal stem cell extracellular vesicles in treating intervertebral disc degeneration,and explore the potential mechanisms of extracellular vesicles and macrophage polarization.Methods A non-contact co culture system for extracellular vesicles,macrophages,and nucleus pulposus cells was established.A control group,a normoxic group,and a hypoxic group were set up.The control group was given macrophages+lipopolysaccharide(LPS)or a co culture system for macrophages and nucleus pulposus cells.The normoxic group was given a co culture system with the addition of normoxic exosomes.The hypoxia group was given a co culture system with the addition of hypoxia induced exosomes.Through quantitative real-time polymerase chain reaction,flow cytometry,β-galactosidase staining and immunofluorescence staining,the effects of normoxic exosomes/hypoxia-induced exosomes on macrophage polarization,as well as on apoptosis and aging of nucleus pulposus cells were measured.The comparison of inter group measurement data was done by t-test.Results Compared with the normoxic group,the expression level of CD86 in the hypoxic group was significantly reduced(2.78±0.31 vs.7.00±0.51,t=8.627,P<0.05),while the expression level of CD163 was significantly increased(19.01±0.38 vs.15.54±0.44,t=9.499,P<0.05).Flow cytometry showed that compared with the normoxic group,the apoptosis rate of nucleus pulposus cells in the hypoxic group was significantly reduced(14.44±0.40 vs.11.16±0.50,t=14.351,P<0.05).The results of galactosidase staining showed that compared with the normoxic group,the aging rate of nucleus pulposus cells in the hypoxic group was significantly reduced(49.75±1.63 vs.59.71±1.63,t=4.425,P<0.05).Immunohistochemical staining confirmed that compared with the normoxic group,the hypoxic group increased the expression of the synthetic gene COL2(1.35±0.03 vs.1.03±0.05,t=6.500,P<0.05)and ACAN(1.66±0.05 vs.1.03±0.05,t=13.141,P<0.05),and decreased the expre

关 键 词：间充质干细胞 外泌体 巨噬细胞 极化 椎间盘退变 

分 类 号：R681.5[医药卫生—骨科学]