骨髓间充质干细胞来源外泌体通过miR-210介导大鼠脊髓损伤后的神经保护作用  

Bone marrow mesenchymal stem cell-derived exosome mediate neuroprotection via miR-210 after spinal cord injury in rats

作　　者：王滋润[1] 梁丽芹 肖成伟 杨晓[1] 邹有策 WANG Zi-run;LIANG Li-qin;XIAO Cheng-wei;YANG Xiao;ZOU You-ce(Department of Orthopedics,Sichuan Academy of Medical Sciences,Sichuan Provincial People's Hospital,Chengdu 610072,China;Department of Surgery,Sichuan Academy of Medical Sciences,Sichuan Provincial People's Hospital,Chengdu 610072,China)

机构地区：[1]四川省医学科学院,四川省人民医院骨科,成都610072 [2]四川省医学科学院,四川省人民医院外科,成都610072

出　　处：《现代免疫学》2022年第1期15-24,36,共11页Current Immunology

摘　　要：为探究抗凋亡miR-210是否参与骨髓间充质干细胞(bone marrow mesenchymal stem cell, BMSC)来源外泌体对脊髓损伤(spinal cord injury, SCI)的保护作用,采用全骨髓贴壁法培养BMSC,并通过ExoQuick外泌体提取试剂盒提取并标记BMSC-外泌体,于透射电子显微镜(transmission electron microscope, TEM)下观察BMSC-外泌体形态。将大鼠随机分为5组:假手术组(Sham)、SCI模型组、SCI+BMSC-外泌体组、SCI+外泌体-miR-210抑制剂组及SCI+外泌体-miR-210抑制剂-NC组。于大鼠SCI造模后1 h尾静脉注射BMSC-外泌体或PBS干预。于损伤后不同时间点评估各组SCI大鼠的行为学评分(Basso-Beattie-Bresnahan, BBB);尼氏(Nissl)染色观察大鼠脊髓神经元的功能状况;RT-qPCR检测BMSC miR-210表达;Western blotting检测损伤脊髓组织Bax、Bcl-2、cleaved caspase-3和caspase-3蛋白的表达水平;TUNEL染色评估脊髓神经元凋亡情况。结果显示,BMSC-外泌体在TEM下呈现类圆形、圆形或茶托状囊泡,直径约40~120 nm;从SCI后第7天(Day 7, D7,后类推)开始,BMSC-外泌体治疗组的BBB评分显著高于SCI模型组(D7、D14、D21和D28,均P<0.05);与BMSC-外泌体组相比,外泌体-miR-210抑制剂组BBB评分显著降低(D7、D14、D21和D28,均P<0.05);与SCI模型组相比,BMSC-外泌体组大鼠TUNEL染色阳性细胞数显著减少(P<0.01),这一抑制作用可被miR-210抑制剂处理逆转(P<0.01);与BMSC-外泌体组相比,外泌体-miR-210抑制剂组脊髓组织细胞Bax/Bcl-2比值和cleaved caspase-3/caspase-3比值显著增加(均P<0.05)。由此,大鼠SCI造模后尾静脉注射BMSC-外泌体可明显减少神经元凋亡并改善大鼠运动功能,其作用机制可能与抗凋亡miR-210的功能发挥有关。To investigate whether anti-apoptotic miR-210 participates in the neuroprotective effect of bone marrow mesenchymal stem cell(BMSC)-derived exosomes in the spinal cord injury(SCI), BMSC was cultured by the whole bone marrow adherent culture. The BMSC-exosomes were extracted and labeled by ExoQuick exosome extraction kit. The morphology of BMSC-exosomes was observed under the transmission electron microscope(TEM). The rats were randomly divided into five groups: sham operation group(Sham), SCI model group, SCI+BMSC-exosomes group, SCI+exosomes-miR-210 inhibitor group and SCI+exosomes-miR-210 inhibitor-NC group. All administrations of BMSC-exosomes or PBS were excuted via tail vein injection at 1 h following SCI surgery. After SCI, rats Basso-Beattie-Bresnahan(BBB) scores in each group were evaluated at different time points;Nissl staining was used to observe rat spinal cord neuron function, and miR-210 expression was analyzed by RT-qPCR;Protein expression levels of Bax, Bcl-2, cleaved caspase-3 and caspase-3 in SCI tissues were analyzed by Western blotting;The apoptosis of spinal cord neurons was examined by TUNEL staining. TEM observation of BMSC-exosomes showed round-like, round or saucer-like vesicles with the diameter ranging from 40 to 120 nm. BBB scores of the BMSC-exosomes treatment group were significantly greater than those of the SCI group starting at Day 7(D7) post-SCI(D7, D14, D21 and D28, all P<0.05);Compared to the BMSC-exosomes treatment group, the BBB scores in the exosome-miR-210 inhibitor group were significantly decreased(D7, D14, D21 and D28, all P<0.05). Compared with SCI model group, the number of TUNEL staining positive cells in BMSC-exosomes treatment group decreased significantly(P<0.01), which could be reversed by miR-210-inhibitor treatment(P<0.01);Furthermore, the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio of exosomes-miR-210-inhibitor group were significantly higher than those of BMSC-exosomes group(all P<0.05). In conclusion, intravenous injection of BMSC-exosomes can signi

关 键 词：骨髓间充质干细胞 外泌体 微小核糖核酸210 脊髓损伤 神经保护 

分 类 号：R651.2[医药卫生—外科学]