中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (6): 1069-1078.doi: 10.4103/1673-5374.250629

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

Pluronic F-127水凝胶慢病毒载体介导缺氧诱导因子1α过表达修复大鼠臂丛神经损伤

  

  • 出版日期:2019-06-15 发布日期:2019-06-15
  • 基金资助:

    国家自然科学基金(81371366)、广东省自然科学基金(2015A030313515)、东莞国际科技合作项目(2013508152010)、中国东莞社会发展重点工程项目(20185071521640)

Effect of lentiviral vector-mediated overexpression of hypoxia-inducible factor 1 alpha delivered by pluronic F-127 hydrogel on brachial plexus avulsion in rats

Tao Wang 1, 2 , Li-Ni Zeng 1 , Zhe Zhu 3 , Yu-Hui Wang 1, 2 , Lu Ding 4 , Wei-Bin Luo 1, 2 , Xiao-Min Zhang 1 , Zhi-Wei He 1 , Hong-Fu Wu 1   

  1. 1 Institute of Stem Cells and Regenerative Medicine, Department of Physiology, Guangdong Medical University, Dongguan, Guangdong Province, China
    2 Department of Surgery, the Third Hospital of Guangdong Medical University (Longjiang Hospital of Shunde District), Foshan, Guangdong Province, China
    3 Hand & Foot Surgery and Reparative & Reconstruction Surgery Center, the Second Hospital of Jilin University, Changchun, Jilin Province, China
    4 Department of Scientific Research Center, the Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, Guangdong Province, China
  • Online:2019-06-15 Published:2019-06-15
  • Contact: Zhi-Wei He, PhD, 467853605@qq.com; Hong-Fu Wu, PhD, hongfuw@126.com.
  • Supported by:

    This work was financially supported by the National Natural Science Foundation of China, No. 81371366 (to HFW); the Natural Science Foundation of Guangdong Province of China, No. 2015A030313515 (to HFW); the Dongguan International Science and Technology Cooperation Project, No. 2013508152010 (to HFW); the Key Project of Social Development of Dongguan of China, No. 20185071521640 (to HFW).

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摘要：

臂丛神经根性撕脱损伤可导致大量运动神经元死亡和严重的运动功能障碍，目前尚无令人满意的修复治疗方法。缺氧诱导因子1α (Hypoxia-inducible factor 1α, HIF-1α)可调控适应缺血-缺氧环境并促进血管新生的相关基因。为此，实验建立SD大鼠臂丛神经根性撕脱再植模型(撕脱C5-C7神经根，仅回植C6神经根)，造模成功后，立即用微量注射器于C6腹侧神经根撕脱回植处进行不同处理后分组：PBS组，NV组(HIF-1α过表达阴性对照慢病毒)，HIF-1α组(HIF-1α过表达慢病毒)，Gel组(PF-127水凝胶)及Gel+HIF-1α组(PF-127水凝胶+HIF-1α过表达慢病毒)。(1)Terzis理毛试验(Terzis grooming test, TGT)检测运动功能恢复情况，与PBS组相比，HIF-1α和Gel+HIF-1α组尤其后者的TGT评分较高；(2)进一步采用电生理，荧光金逆行示踪和免疫荧光染色方法检测神经通路重建情况及脊髓神经元、新生血管和肌肉运动终板的变化情况，与PBS组相比HIF-1α和Gel + HIF-1α组肌皮神经动作电位潜伏期明显缩短、荧光金阳性细胞数及ChAT阳性神经元、撕脱周围脊髓新生血管标志物CD31阳性细胞数、肱二头肌α-BtX运动终板形态数量明显增加；(3)上述结果说明，利用水凝胶递送HIF-1α过表达慢病毒载体，可有效促进大鼠臂丛根性撕脱损伤的神经修复和运动功能的恢复。

orcid: 0000-0002-3914-4752 (Zhi-Wei He)
           0000-0002-1115-3681 (Hong-Fu Wu)

关键词: 周围神经损伤, 臂丛神经撕脱, 缺氧, 缺血, 缺氧诱导因子1&alpha, 过表达, Pluronic F-127水凝胶, 运动神经元, 轴突再生, 血管新生, 神经再生

Abstract:

Brachial plexus avulsion often results in massive motor neuron death and severe functional deficits of target muscles. However, no satisfactory treatment is currently available. Hypoxia-inducible factor 1α is a critical molecule targeting several genes associated with ischemia-hypoxia damage and angiogenesis. In this study, a rat model of brachial plexus avulsion-reimplantation was established, in which C5–7 ventral nerve roots were avulsed and only the C6 root reimplanted. Different implants were immediately injected using a microsyringe into the avulsion-reimplantation site of the C6 root post-brachial plexus avulsion. Rats were randomly divided into five groups: phosphate-buffered saline, negative control of lentivirus, hypoxia-inducible factor 1α (hypoxia-inducible factor 1α overexpression lentivirus), gel (pluronic F-127 hydrogel), and gel + hypoxia-inducible factor 1α (pluronic F-127 hydrogel + hypoxia-inducible factor 1α overexpression lentivirus). The Terzis grooming test was performed to assess recovery of motor function. Scores were higher in the hypox¬ia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups (in particular the gel + hypoxia-inducible factor 1α group) compared with the phosphate-buffered saline group. Electrophysiology, fluorogold retrograde tracing, and immunofluorescent staining were further performed to investigate neural pathway reconstruction and changes of neurons, motor endplates, and angiogenesis. Compared with the phosphate-buffered saline group, action potential latency of musculocutaneous nerves was markedly shortened in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Meanwhile, the number of fluorogold-positive cells and ChAT-positive neurons, neovascular area (labeled by CD31 around avulsed sites in ipsilateral spinal cord segments), and the number of motor endplates in biceps brachii (identified by α-bungarotoxin) were all visibly increased, as well as the morphology of motor endplate in biceps brachil was clear in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Taken together, delivery of hypoxia-inducible factor 1α overexpression lentiviral vectors mediated by pluronic F-127 effectively promotes spinal root regeneration and functional recovery post-brachial plexus avulsion. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, China.

Key words: nerve regeneration, peripheral nerve injury, brachial plexus avulsion, hypoxia, ischemia, hypoxia-inducible factor 1&alpha, overexpression, pluronic F-127, motor neurons, axonal regeneration, angiogenesis, neural regeneration