骨髓间充质干细胞来源外泌体改善脊髓损伤后血脊髓屏障的完整性

Exosomes derived from bone marrow mesenchymal stem cells improve the integrity of the blood-spinal cord barrier after spinal cord injury

背景:研究发现,干细胞衍生的外泌体可通过多种途径促进脊髓损伤后运动功能的恢复。目的:研究骨髓间充质干细胞来源外泌体是否通过减轻血脊髓屏障的破坏来促进脊髓损伤后运动功能的恢复。方法:60只SD大鼠随机分成假手术组、模型组和外泌体组(n=20),采用改良Allen’s法建立大鼠脊髓损伤模型,外泌体组在损伤后30 min,1 d分别经尾静脉注射200μL骨髓间充质干细胞来源外泌体,在损伤后第3天,通过伊文思蓝染色观察血脊髓屏障通透性,Western blot检测基质金属蛋白酶9及紧密连接蛋白claudin-5,Occludin,ZO-1的表达,明胶酶谱法检测基质金属蛋白酶9的活性,免疫荧光检测中性粒细胞浸润情况,苏木精-伊红染色观察脊髓损伤形态学变化;在损伤后第1,3,5,7,10,14,21天,采用BBB评分量表评价运动功能恢复情况。结果与结论:①外泌体组大鼠的BBB评分在第10,14,21天显著高于模型组(P<0.05),苏木精-伊红染色结果显示外泌体组相比于模型组损伤区明显缩小(P<0.05);②外泌体治疗后血脊髓屏障染料渗出量显著减少(P<0.05),紧密连接蛋白claudin-5、Occludin、ZO-1的表达相比于模型组显著增加(P<0.05);③外泌体抑制基质金属蛋白酶9的表达和活性(P<0.05);④损伤后第3天于病变部位观察到髓过氧化物酶阳性的中性粒细胞浸润,外泌体治疗能显著减少中性粒细胞的浸润(P<0.05);⑤结果表明,骨髓间充质干细胞来源外泌体通过下调基质金属蛋白酶9的表达和活性来减少紧密连接蛋白的降解,从而减轻血脊髓屏障的破坏和随后的中性粒细胞浸润,进而促进脊髓损伤后运动功能的恢复。

BACKGROUND:The studies reveal that stem cells-derived exosomes promote locomotor recovery after spinal cord injury.OBJECTIVE:To investigate whether the bone marrow mesenchymal stem cells-derived exosomes promote locomotor recovery via inhibiting the damage of the blood-spinal cord barrier.METHODS:Sixty Sprague-Dawley rats were randomly divided into sham operation group,model group,and exosome group(n=20).The rat models of spinal cord injury were established by modified Allen’s method.In the exosome group,200μL of bone marrow mesenchymal stem cells-derived exosomes were injected through the tail vein at 30 minutes and 1 day after injury.On the third day after injury,the permeability of the blood-spinal cord barrier was observed.Western blot assay was used to detect the expression of matrix metalloproteinase-9 and claudin-5,Occludin and ZO-1.Gelatin zymography was used to detect the activity of matrix metalloproteinase-9.Immunofluorescence was used to detect the infiltration of neutrophils.Hematoxylin-eosin staining was used to observe the morphological changes of spinal cord injury.At 1,3,5,7,10,14,and 21 days after injury,the Basso-Beattie-Bresnahan rating scale was used to evaluate the recovery of motor function.RESULTS AND CONCLUSION:(1)The Basso-Beattie-Bresnahan scores of rats treated with exosome were significantly higher than these spinal cord injury rats at 10,14,and 21 days after spinal cord injury(P<0.05).The results of hematoxylin-eosin staining showed that the damage area in the exosome group was significantly reduced compared to the model group(P<0.05).(2)Exosome treatment significantly reduced the amount of Evans Blue dye extravasation(P<0.05).Compared with the model group,the expression levels of tight junction protein,including claudin-5,Occludin and ZO-1,were significantly increased in the exosome group(P<0.05).(3)Exosome inhibited matrix metalloproteinase-9 expression and activity(P<0.05).(4)Infiltrated myeloperoxidase-positive neutrophils were observed at the lesion site of injured spinal cord at 3 days after spinal cord injury.The exosome treatment significantly inhibited the infiltration of neutrophils(P<0.05).(5)The results suggested that bone marrow mesenchymal stem cells-derived exosome improved functional recovery after spinal cord injury in part by inhibiting blood-spinal cord barrier disruption and the subsequent infiltration of neutrophils through reducing the degradation of tight junction proteins by inhibiting the expression and activity of matrix metalloproteinase-9.