骨髓间充质干细胞外泌体可减少脊髓损伤后A1型星形胶质细胞的活化

Bone marrow mesenchymal stem cell-derived exosomes reduce the activation of type A1 astrocytes after spinal cord injury

背景:课题组前期研究发现,骨髓间充质干细胞来源外泌体可减少脊髓损伤区域反应性星形胶质细胞的数量,有利于损伤后运动功能的改善。目的:探索骨髓间充质干细胞外泌体对脊髓损伤后A1型星形胶质细胞活化的影响及其在损伤后修复中的作用。方法:培养SD大鼠骨髓间充质干细胞,利用超速离心法提取细胞上清液中的外泌体并进行鉴定。将80只成年雄性SD大鼠随机分为假手术组、脊髓损伤模型组、骨髓间充质干细胞组、骨髓间充质干细胞外泌体组,每组20只。除假手术组外,其余3组均使用Allen法制备脊髓损伤模型,术后2 h通过尾静脉注射等体积的PBS、骨髓间充质干细胞悬液、骨髓间充质干细胞外泌体悬液,每隔2d注射1次,共注射3次。脊髓损伤术前、术后1,3,7,14,28 d采用BBB评分法评估后肢运动功能;术后7 d通过苏木精-伊红染色、透射电镜观察脊髓组织形态,免疫荧光检测A1型星形胶质细胞数量、NeuN阳性神经元数量、caspase-3阳性凋亡细胞数量,ELISA法检测脊髓组织肿瘤坏死因子α、白细胞介素1α水平,Western blot检测脊髓组织C3、GFAP的表达水平。结果与结论:①骨髓间充质干细胞外泌体在透射电镜下呈茶托状,表达外泌体标志CD63、CD9;②骨髓间充质干细胞外泌体减少脊髓组织炎症细胞浸润、减轻髓鞘脱失、轴突退变,降低炎症因子肿瘤坏死因子α、白细胞介素1α水平和C3的表达水平,降低A1型星形胶质细胞数量,减少细胞凋亡,提高残余神经元数量,改善肢体运动功能;③以上结果提示,骨髓间充质干细胞外泌体能减少脊髓A1型星形胶质细胞的活化,促进脊髓损伤后运动功能恢复。

BACKGROUND:Previous studies found that exosomes from bone marrow mesenchymal stem cells could reduce the number of reactive astrocytes after spinal cord injury,thereby improving post-injury motor function.OBJECTIVE:To explore the role of bone marrow mesenchymal stem cell-derived exosomes in the activation of A1 astrocytes as well as in the repair of spinal cord injury.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawely rats were cultured,and then the exosomes from the cell supernatant were extracted and identified by ultracentrifugation.Eighty adult male Sprague-Dawley rats were randomly divided into sham operation group,spinal cord injury group(model group),bone marrow mesenchymal stem cell group(cell transplantation group)and exosome group(n=20/group).Except the sham operation group,an animal model of spinal cord injury was made in all the groups by Allen method.Two hours postoperatively,the equal volume of PBS,bone marrow mesenchymal stem cell suspension or bone marrow mesenchymal stem cell-derived exosome suspension was injected through the tail vein respectively,every 3 days,for three sessions.Basso,Beattie and Bresnahan scoring for motor function of the hind limb was performed preoperatively and and 1,3,7,14,28 days postoperatively.Hematoxylin-eosin staining and transmission electron microscope were used to observe the morphology of spinal cord tissue at 7 days postoperatively.Immunofluorescence staining was used to detect the number of A1 astrocytes,NeuN positive neurons and Caspase-3 positive apoptotic cells.ELISA assay was used to detect the levels of tumor necrosis factor-αand interleukin-1αin the spinal cord tissue.Western blot assay was used to determine relative protein expression levels of C3 and GFAP.RESULTS AND CONCLUSION:Exosomes from bone marrow mesenchymal stem cells showed a saucer-like appearance under transmission electron microscope,expressing exosome markers CD63 and CD9.The exosomes reduced inflammatory cell infiltration of spinal cord tissue,reduced myelin degeneration and axonal degeneration,reduced the expression levels of tumor necrosis factor-α,interleukin-1αand C3,reduced the number of A1 astrocytes,and reduced cell apoptosis,but increased the number of residual neurons,and improved limb motor function.To conclude,exosomes from bone marrow mesenchymal stem cells can reduce the activation of type A1 astrocytes in the spinal cord and promote the recovery of motor function after spinal cord injury.