外胚层间充质干细胞来源的外泌体通过控制炎症和氧化损伤减少M1型小胶质细胞并促进H2O2处理后PC12细胞的存活  

Exosomes from ectoderm mesenchymal stem cells inhibits lipopolysaccharide-induced microglial M1 polarization and promotes survival of H2O2-exposed PC12 cells by suppressing inflammatory response and oxidative stress

作　　者：孙晓鹏 史航 张磊 刘中 李克威 钱玲玲 朱星宇 杨康佳 付强 丁华[1] SUN Xiaopeng;SHI Hang;ZHANG Lei;LIU Zhong;LI Kewei;QIAN Lingling;ZHU Xingyu;YANG Kangjia;FU Qiang;DING Hua(Department of Orthopedics,Affiliated People's Hospital of Jiangsu University,Zhenjiang 212000,China;Department of Orthopedics,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China;School of Medicine,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学附属人民医院骨科,江苏镇江212000 [2]上海交通大学医学院附属第一人民医院骨科,上海200080 [3]江苏大学医学院,江苏镇江212013

出　　处：《南方医科大学学报》2024年第1期119-128,共10页Journal of Southern Medical University

基　　金：江苏省卫健委项目(Z2020058);江苏大学临床医学科技发展基金项目(JLY2021012);镇江市卫健委“金山医者”人才项目(2022-JSYZ-1);江苏省高层次卫生人才“六个一工程”拔尖人才科研项目(LGY2020065);2021年江苏省研究生实践创新计划(SJCX21-1725)。

摘　　要：目的探究外胚层间充质干细胞来源的外泌体(EMSCs-exo)对脊髓继发性损伤的潜在修复作用。方法从大鼠鼻黏膜中分离培养EMSCs,并通过免疫荧光染色鉴定。采用超速离心法获取EMSCs-exo,并通过透射电镜、纳米颗粒跟踪分析(NTA)和Westernblot进行鉴定。利用差速贴壁法纯化小胶质细胞,并通过免疫荧光染色鉴定。根据BCA法测得的蛋白浓度对EMSCs-exo进行定量分析。设置对照组、含100μg/L脂多糖(LPS)的组和含LPS及37.5mg/L或75mg/LEMSCs-exo的组对小胶质细胞进行处理。设置对照组、含400μmol/LH2O2的组和含H2O2及37.5mg/L或75mg/LEMSCs-exo的组对PC12细胞进行处理。通过Western blot和qRT-PCR测定小胶质细胞各组Arg1和iNOS蛋白和mRNA表达量。通过酶联免疫吸附实验测定上清中IL-6、IL-10和IGF-1的浓度。通过CCK-8和AnnexinV-FITC/PI凋亡检测试剂盒检测PC12细胞各组的活力和凋亡情况。结果免疫荧光染色显示EMSCs高表达标志物Nestin、CD44、CD105、Vimentin。透射电镜显示EMSCs-exo呈典型的杯状结构,NTA显示其平均粒径为142nm,Westernblot显示其表达外泌体标志蛋白CD63、CD81、TSG101,不表达Vimentin。当小胶质细胞在LPS环境中时,75mg/L的EMSCs-exo能够有效提高其Arg1蛋白量、降低iNOS蛋白量(P<0.05),且相比于37.5mg/L的浓度,其更能有效提高其Arg1mRNA水平和IGF-1、IL-10(P<0.05)的生成,降低iNOSmRNA水平和IL-6的生成(P<0.05);也更有效促进H2O2环境中PC12细胞的存活,降低凋亡率(P<0.05)。结论75mg/L的EMSCs-exo在体外有效减少M1型小胶质细胞比例,减轻氧化应激下的神经元凋亡,促进神经元存活,因此在控制脊髓继发性损伤中具有一定应用潜能。Objective To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells(EMSCs-exo)for repairing secondary spinal cord injury.Methods EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa,identified by transmission electron microscope,nanoparticle tracking analysis(NTA),and Western blotting,and quantified using the BCA method.Neonatal rat microglia purified by differential attachment were induced with 100μg/L lipopolysaccharide(LPS)and treated with 37.5 or 75 mg/L EMSCs-exo.PC12 cells were exposed to 400μmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L.The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT-PCR,and the concentrations of IL-6,IL-10,and IGF-1 in the supernatants were measured with ELISA.The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry.Results The isolated rat EMSCs showed high expressions of nestin,CD44,CD105,and vimentin.The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63,CD81,and TSG101 but not vimentin.In LPS-treated microglia,EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression(P<0.05).EMSCs-exo treatment at 75 mg/L,as compared with the lower concentration at 37.5 mg/L,more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia,and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells(P<0.05).Conclusion EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival,suggesting its potential in controlling secondary spinal cord injury.

关 键 词：外胚层间充质干细胞 外泌体 小胶质细胞 炎症 氧化应激 脊髓继发性损伤 

分 类 号：R651.2[医药卫生—外科学]