骨髓间充质干细胞来源的外泌体用于大鼠脊髓损伤修复的初步探索

目的:研究大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分泌的外泌体(exosomes)对脊髓损伤大鼠行为学、损伤处活化星形胶质细胞和残余神经元数量的影响,初步探索BMSCs-exosomes作为脊髓损伤的细胞替代疗法的可行性。方法 :采用全骨髓培养法培养BMSCs,流式细胞术鉴定其表面标志CD90和CD34。收集BMSCs上清液,超速离心法提取并标记外泌体,电镜观察其结构,Western blot法检测其CD63和CD9的表达水平。将外泌体作用于脊髓损伤大鼠,于损伤后各时点进行后肢运动功能评价,并于预定时点处死大鼠取损伤脊髓段,尼氏染色法观察损伤脊髓形态,免疫荧光法检测反应性星形胶质细胞和残余神经元的数量。结果:BMSCs分泌的外泌体电镜下呈"茶托状"结构,Western blot法检测其阳性表达CD63和CD9。脊髓损伤后14d和28 d治疗组神经功能有一定程度改善,与损伤组间差异有统计学意义(P0.05)。损伤后14 d治疗组较之损伤组的尼氏体结构更完整、分布更匀称,崩解凝聚减少。治疗组损伤后7 d、14 d和28 d反应性星形胶质细胞数量较损伤组明显降低(P0.05)。损伤后7 d、14 d和28 d治疗组残余神经元数量高于相应时点损伤组(P0.05)。结论:BMSCs分泌的外泌体可减少脊髓损伤区域反应性星形胶质细胞的数量,减少神经元的死亡,有利于损伤后后肢运动功能改善。

Preliminary study of bone marrow mesenchymal stem cell-derived exosomes in repair of spinal cord injury in rats

AIM:To study the influence of bone marrow mesenchymol stem cell-drived exosomes (BMSC-exosomes) on hindlimb activity, and the numbers of reactive astrocytes and residual neurons in spinal cord injury (SCI) rats. METHODS:BMSCs were cultured using the whole bone marrow adherent culture method and surface markers CD90 and CD34 were verified by flow cytometry. Exosomes were isolated by ultracentrifugation and the morphology of exosomes was observed under transmission electron microscope. The protein markers CD63 and CD9 were verified by Western blot. After exosomes were applied to SCI rats, the Basso, Beattie and Bresnahan locomotor rating scale score, the Nissl staining of the lesion site, and the numbers of reactive astrocytes and residual neurons were assessed at various time points. RESULTS:Transmission electron microscopic observation revealed the presence of saucer-shaped vesicles. BMSC-exosomes were found to express high levels of CD63 and CD9. Compared with injury group, significant improvement of hindlimb activity scores from day 14 after injury in treatment group was observed (P<0.05), and less reactive astrocytes and more residual neurons from day 7 after injury were also observed (P<0.05). CONCLUSION:BMSC-exosomes inhibit reactive astrocytes and death of neurons, and improve hindlimb activity in the rats after SCI.