嗅鞘细胞外泌体通过促进血管生成改善大鼠脊髓损伤的短期疗效分析

Short-term effects of olfactory ensheathing cell-derived exosomes in improving spinal cord injury in rats by promoting angiogenesis

摘要

目的 分析嗅鞘细胞来源的外泌体（OECs-Exos）对脊髓损伤（SCI）后血管生成及神经功能的影响。方法 从嗅鞘细胞培养液中提取并鉴定OECs-Exos，分别通过细胞实验和动物实验进行研究。选用大鼠微血管内皮细胞，分为对照组和OECs-Exos组，培养基中分别加入50 μL磷酸盐缓冲生理盐水（PBS）和50 μL OECs-Exos。采用EdU增殖、Transwell迁移及管形成实验观察内皮细胞血管生成活性。45只SD雌性大鼠随机分为假手术组、SCI组和OECs-Exos组。SCI组和OECs-Exos组行脊髓损伤模型制备，术后30 min及1、2 d，分别经尾静脉注射200 μL PBS和200 μL OECs-Exos（200 μg/mL）；假手术组仅行椎板切除，经尾静脉注射200 μL PBS。通过巴索-比蒂-布雷斯纳汉（BBB）评分评估大鼠术前及术后后肢运动情况。采用分化簇（CD）31免疫荧光染色观察术后第7天脊髓损伤区域血管生成情况。术后第14天，通过尼氏染色、生长相关蛋白（GAP）43免疫荧光染色观察脊髓损伤区域神经元存活量和新生轴突数量。结果 细胞实验显示，OECs-Exos可被内皮细胞摄取，OECs-Exos显著促进内皮细胞增殖、迁移及血管形成。CD31免疫荧光染色显示，OECs-Exos显著促进损伤区域血管内皮细胞生成；尼氏染色证实OECs-Exos显著减少损伤区域的神经元损伤；GAP43免疫荧光染色显示，OECs-Exos显著增加损伤区域的轴突数量；BBB评分结果证实OECs-Exos改善了大鼠SCI后7~28 d的运动功能。结论 OECs-Exos可能通过促进血管生成，改善局部微环境，增强神经元保护和轴突再生，减少继发性损伤，有利于大鼠运动功能的恢复。

Abstract

Objective To investigate the effects of olfactory ensheathing cell-derived exosomes (OECs-Exos) on angiogenesis and neurological function after spinal cord injury (SCI). Methods OECs-Exos were extracted and identified from the culture medium of olfactory ensheathing cells, and studied by in vitro cell experiments and in vivo animal experiments, respectively. Rat microvascular endothelial cells were divided into a control group and an OECs-Exos group, and 50 μL PBS and 50 μL OECs-Exos were added to the medium, respectively. The angiogenic activity of endothelial cells was observed by EdU proliferation assay, Transwell migration assay and tube formation assay. Forty-five female Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a SCI group and an OECs-Exos group. The SCI model was established in the SCI group and the OECs-Exos group. At 30 min, 1 d and 2 d after operation, 200 μL phosphate-buffered saline (PBS) and 200 μL OECs-Exos (200 μg/mL) were injected via the tail vein, respectively. The sham-operated group only received laminectomy and was injected with 200 μL PBS via the tail vein. Hindlimb motor function in rats was evaluated before and after surgery using the Basso-Beattie-Bresnahan (BBB) score. Angiogenesis in the injured spinal cord was observed by cluster of differentiation (CD) 31 immunofluorescence staining on the 7th day after surgery. On the 14th day after surgery, Nissl staining and growth associated protein (GAP) 43 immunofluorescence staining were used to observe the number of surviving neurons and regenerating axons in the injured spinal cord. Results Cell experiments showed that OECs-Exos could be taken up by endothelial cells, and OECs-Exos significantly promoted endothelial cell proliferation, migration and angiogenesis. CD31 immunofluorescence staining showed that OECs-Exos significantly promoted angiogensis in the injured area. Nissl staining confirmed that OECs-Exos significantly reduced neuronal damage in the injured area. GAP43 immunofluorescence staining showed that OECs-Exos significantly increased the number of axons in the injured area. BBB scores confirmed that OECs-Exos improved motor function of rats from 7 to 28 days after SCI. Conclusion OECs-Exos may promote angiogenesis, improve the local microenvironment, enhance neuronal protection and axonal regeneration, reduce secondary injury, and thereby facilitate the recovery of motor function in rats.

关键词

外泌体 / 新生血管化，生理性 / 内皮细胞 / 脊髓损伤

Key words

Exosomes / Neovascularization, physiologic / Endothelial cells / Spinal cord injuries

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基金资助

河南省医学科技攻关计划项目(LHGJ20230473)