中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2017, Vol. 12 ›› Issue (9): 1507-1518.doi: 10.4103/1673-5374.215263

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

甲氨蝶呤联合甲基泼尼松龙治疗脊髓损伤大鼠运动功能恢复和316个差异基因表达谱的变化

  

  • 收稿日期:2017-08-12 出版日期:2017-09-15 发布日期:2017-09-15
  • 基金资助:

    国家自然科学基金（30960448）；江西省自然科学基金（20142bab205023）

Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury

Jian-tao Liu1, Si Zhang1, Bing Gu1, Hua-nan Li2, Shuo-yu Wang2, Shui-yin Zhang1   

  1. 1 Jiangxi Key Laboratory of Bioprocess Engineering, Jiangxi Science & Technology Normal University, Nanchang, Jiangxi Province, China
    2 Department of Spine Surgery, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi Province, China
  • Received:2017-08-12 Online:2017-09-15 Published:2017-09-15
  • Contact: Bing Gu, Ph.D.,bguemory@hotmail.com.
  • Supported by:

    This research was supported by the National Natural Science Foundation of China, No. 30960448; the Natural Science Foundation of Jiangxi Province, No. 20142BAB205023; the Ph.D. Start-up Fund of Natural Science Foundation of Jiangxi Science & Technology Normal University in China, No. 3000990122.

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摘要：

甲基强的松龙是目前治疗脊髓损伤临床主要药物，但高剂量甲基强的松龙会增加感染性疾病等并发症的发生。甲氨蝶呤具有抗炎活性和免疫抑制功效，可减轻脊髓损伤后的炎症反应。为分析甲氨蝶呤与甲基强的松龙联合治疗脊髓损伤的分子机制，以及基因表达谱的变化。实验设计采用PinPointTM精密皮质撞击器制备脊髓挫伤大鼠模型，先于损伤后30 min静脉注射甲基强的松龙30 mg/kg，再于损伤后1 d皮下注射甲氨蝶呤0.3 mg/kg，1次/d，连续2周。TreadScan步态分析系统检测发现损伤后4和8周，甲氨蝶呤和甲基泼尼松龙联合干预的脊髓损伤大鼠后肢摆动时间、跨步时间、最小纵向偏差、瞬间跑速、脚印面积和正常步序均显著改善。采用Solexa高通量测序技术分析基因差异表达情况，与单独甲基强的松龙干预比较，甲氨蝶呤和甲基泼尼松龙联合干预后脊髓损伤大鼠脊髓组织可见316个差异表达基因，上调基因的275个功能主要涉及促神经恢复、抗氧化、抗炎及抗凋亡方面，下调基因的41个功能主要涉及促炎和促凋亡方面。结果可以说明，甲氨蝶呤和甲基泼尼松龙联合干预能弥补单一甲氨蝶呤治疗的不足，在一定程度上抑制炎性细胞因子的活性，增强抗氧化和抗凋亡的作用，提高神经保护效果，在此过程中有316个差异表达基因发挥了重要的作用。

orcid:0000-0001-7900-6605(Bing Gu)

关键词: 脊髓损伤, 甲氨蝶呤, 甲基强的松龙, 步态, 基因表达谱, 炎症反应, 氧化应激, 凋亡, 神经修复, Solexa 基因测序, 继发性损伤

Abstract:

Methylprednisolone is a commonly used drug for the treatment of spinal cord injury, but high doses of methylprednisolone can increase the incidence of infectious diseases. Methotrexate has anti-inflammatory activity and immunosuppressive effects, and can reduce inflammation after spinal cord injury. To analyze gene expression changes and the molecular mechanism of methotrexate combined with methylprednisolone in the treatment of spinal cord injury, a rat model of spinal cord contusion was prepared using the PinPoint™ precision cortical impactor technique. Rats were injected with methylprednisolone 30 mg/kg 30 minutes after injury, and then subcutaneously injected with 0.3 mg/kg methotrexate 1 day after injury, once a day, for 2 weeks. TreadScan gait analysis found that at 4 and 8 weeks after injury, methotrexate combined with methylprednisolone significantly improved hind limb swing time, stride time, minimum longitudinal deviation, instant speed, footprint area and regularity index. Solexa high-throughput sequencing was used to analyze differential gene expression.Compared with methylprednisolone alone, differential expression of 316 genes was detected in injured spinal cord treated with methotrexate and ethylprednisolone. The 275 up-regulated genes were mainly related to nerve recovery, anti-oxidative, anti-inflammatory and anti-apoptotic functions, while 41 down-regulated genes were mainly related to proinflammatory and pro-apoptotic functions. These results indicate that methotrexate combined with methylprednisolone exhibited better effects on inhibiting the activity of inflammatory cytokines and enhancing antioxidant and anti-apoptotic effects and thereby produced stronger neuroprotective effects than methotrexate alone. The 316 differentially expressed genes play an important role in the above processes.
 

Key words: nerve regeneration, spinal cord injury, methotrexate, methylprednisolone, gait, gene expression profile, inflammation, oxidative stress, apoptosis, nerve repair, Solexa gene sequencing, secondary lesion, neural regeneration