骨髓间充质干细胞来源外泌体对大鼠脊髓损伤的改善作用及对脊髓组织胞浆型磷脂酶A2、水通道蛋白4和水通道蛋白9的影响实验研究

Improvement effects of BMSCs-derived exosomes on spinal cord injury in rats and its effects on cytosolic phospholipase A2,aquaporin 4 and aquaporin 9 in spinal cord tissues

目的:探讨骨髓间充质干细胞(BMSCs)来源外泌体对大鼠脊髓损伤(SCI)的改善作用及对脊髓组织胞浆型磷脂酶A2(cPLA2)、水通道蛋白4(AQP4)和AQP9的影响。方法:培养BMSCs,提取外泌体并进行鉴定。选择成年雄性SD大鼠30只,分为假手术组、模型组和外泌体组,各10只。采用Allen法制作大鼠脊髓损伤模型,外泌体组经尾静脉注射外泌体,假手术组和模型组大鼠尾静脉注射等体积PBS缓冲液。各组大鼠每隔2 d注射1次,共注射3次。采用脊髓损伤行为学(BBB)评分评估干预前后各组大鼠四肢运动功能。采用HE染色观察脊髓组织结构。采用原位末端脱氧核苷酸转移酶标记法(TUNEL)检测神经细胞凋亡情况。采用实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹法(Western blot)检测脊髓组织PLA2、AQP4、AQP9 mRNA及蛋白表达。结果:BMSCs生长状态良好,外泌体表面标志蛋白CD63、Alix呈阳性表达。干预后3、7 d,外泌体组BBB评分高于模型组(均P<0.05)。假手术组脊髓组织无损伤坏死及出血,结构完整,灰质白质边界清晰,细胞排列有序,细胞核形态正常;模型组大鼠脊髓结构紊乱,大量炎性细胞浸润,伴有囊性腔和空泡,神经元和细胞水肿,神经元数量明显减少;外泌体组脊髓损伤区组织结构紊乱、炎性细胞浸润减轻,脊髓空洞小,损伤恢复较好。假手术组脊髓组织中凋亡细胞较少。模型组细胞凋亡率高于假手术组,而外泌体组细胞凋亡率低于模型组(均P<0.05)。模型组脊髓组织cPLA2、AQP4、AQP9 mRNA及蛋白表达高于假手术组,而外泌体组脊髓组织cPLA2、AQP4、AQP9 mRNA及蛋白表达低于模型组(均P<0.05)。结论:BMSCs来源外泌体对脊髓损伤有一定改善作用,可下调cPLA2、AQP4、AQP9 mRNA及蛋白的表达。

Objective:To investigate the improvement effects of bone marrow mesenchymal stem cells(BMSCs)-derived exosomes on the spinal cord injury(SCI)in rats and its effects on cytoplasmic phospholipase A2(cPLA2)and aquaporin 4(AQP4)and AQP9 in spinal cord tissues.Methods:BMSCs were cultured,and exosomes were extracted and identified.Thirty adult male SD rats were selected and divided into sham operation group,model group and exosome group with 10 rats each.The rat SCI model was established by Allen method.The exosome group was injected with exosomes through the tail vein,and the sham operation group and the model group were injected into the tail vein with equal volume of PBS,once every 2 days for a total of 3 times.BBB score was used to evaluate the limb motor function of rats in each group before and after intervention.The spinal cord tissue structure was observed by HE staining,and the apoptosis of nerve cells was detected by TUNEL,and the mRNA and protein expressions of cPLA2,AQP4 and AQP9 in spinal cord tissues were detected by RT-qPCR and Western blot.Results:BMSCs grew well,and the exosome surface marker proteins CD63 and Alix were positively expressed.At 3 and 7 days after intervention,BBB scores in the exosome group were higher than those in the model group(all P<0.05).In sham operation group,the spinal cord tissue showed no injury,necrosis or bleeding,complete structure,clear gray matter and white matter boundary,orderly cell arrangement,and normal nucleus morphology.In the model group,the spinal cord structure of rats was disturbed,inflammatory cell infiltration was obvious,accompanied by cystic cavity and vacuole,neuron and cell edema,and the number of neurons was significantly reduced.In the exosome group,the tissue structure of the injured area of spinal cord was disturbed,the inflammatory cell infiltration was reduced,the spinal cavity was small,and the injury recovery was better.There were fewer apoptotic cells in the spinal cord tissue in the sham operation group.The apoptosis rate of the model group was higher than that of the sham operation group,and the apoptosis rate of the exosome group was lower than that of the model group(all P<0.05).The mRNA and protein expressions of cPLA2,AQP4 and AQP9 in the spinal cord of the model group were higher than those of the sham operation group,and the mRNA and protein expressions of cPLA2,AQP4 and AQP9 in the spinal cord of the exosome group were lower than those of the model group(all P<0.05).Conclusion:BMSCs-derived exosomes can improve spinal cord injury,and down-regulates the mRNA and protein expressions of cPLA2,AQP4,and AQP9.