中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (7): 1163-1168.doi: 10.3969/j.issn.1673-8225.2011.07.006

• 神经组织构建 nerve tissue construction • 上一篇    下一篇

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤

李培建1，李兵仓2   

  1. 1解放军北京军区总医院神经外科，北京市  100700
    2 解放军第三军医大学野战外科研究所六室，重庆市  400042
  • 收稿日期:2010-08-13 修回日期:2010-09-30 出版日期:2011-02-12 发布日期:2011-02-12
  • 通讯作者: 李兵仓，博士，教授，研究员，解放军第三军医大学野战外科研究所六室，重庆市 400042 bcli1118@yahoo.com
  • 作者简介:李培建☆，男，1965年生，河南省辉县人，汉族，2002年解放军第三军医大学毕业，博士，副主任医师，主要从事神经系统损伤的基础和临床研究。 Lpj6507@yahoo.com.cn
  • 基金资助:

    国家重点基础研究发展规划专项资助项目(G1999054206)。

Adenovirus-mediates gene transfer of brain-derived neurotrophic factor for repairing sciatic nerve injury

Li Pei-jian1, Li Bing-cang2   

  1. 1Department of Neurosurgery, Beijing Military General Hospital of Chinese PLA, Beijing  100700, China
    2Six Room, Research Institute of Surgery, Third Military Medical University of Chinese PLA, Chongqing  400042, China
  • Received:2010-08-13 Revised:2010-09-30 Online:2011-02-12 Published:2011-02-12
  • Contact: Li Bing-cang, Doctor, Professor, Researcher, Six Room, Research Institute of Surgery, Third Military Medical University of Chinese PLA, Chongqing 400042, China bcli1118@yahoo.com
  • About author:Li Pei-jian☆, Doctor, Associate chief physician, Department of Neurosurgery, Beijing Military General Hospital of Chinese PLA, Beijing 100700, China Lpj6507@yahoo.com.cn
  • Supported by:

    the National Basic Research Program of China No. G1999054206*

PDF

399

被引次数

5

摘要：

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。
目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor，BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达，以及脊髓前角运动神经元的存活和神经生长情况。
方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经，AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液，BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3，7，14 d，1，2，4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达，并观察损伤坐骨神经的组织学及超微结构改变，再生的神经元及有髓神经纤维数目和髓鞘厚度。
结果与结论：术后3，7，14 d及1个月时，AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实，BDNF基因转染后，脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达，并通过轴突逆行转运到了相应的脊髓神经元，不仅能促进损伤神经纤维再生，也能保护损伤的脊髓神经元。

关键词: 坐骨神经损伤, 重组腺病毒, 脑源性神经营养因子, 基因转染, 免疫组织化学, 原位分子杂交, 神经再生

Abstract:

BACKGROUND: How to accelerate injury repair and regeneration following peripheral nerve injury is the research focus. Gene therapy may be the possible treatment for this problem. 
OBJECTIVE: To observe the expression of the brain-derived neurotrophic factor (BDNF) gene after microinjected adenovirus-mediated gene transfer of BDNF (AxCA-BDNF) to the sciatic nerve for peripheral nerve regeneration.
METHODS: Based on silicone tube graft as a support to bridge adult rat sciatic nerve gaps, Wistar rat were microinjected recombinant adenovirus vector of BDNF (AxCA-BDNF), BDNF and simple injection of virus buffer to the sciatic nerve respectively. With the methods of in situ hybridization and immunocytochemistry, the BDNF gene expression was certified, the number of the new nerve fibers and motoneurons in anterior horn of the spinal cord were calculated, and the myelin sheath thickness of the new nerve fibers was measured at 3, 7, 14 days and 1, 2, 4 months after operation.
RESULTS AND CONCLUSION: Compared with the BDNF and control group, the expression of the BDNF gene in the proximal end, distal end and spinal cord (L3-6) of injured sciatic nerve were obviously higher than that of the BDNF and control groups    (P < 0.01). The result of retrograde axonal transport of HRP tracer indicated the survival neurons, regenerated nerve fibers, thickness of myelin sheath, as well as the re-formation of nerve connection of the AxCA-BDNF group were superior to the control group(P < 0.01). The results demonstrated that exogenous BDNF gene and its express proteins were uptaken to the spinal cord motoneurons through retrograde axonal transport. Gene therapy for sciatic nerve injury of adult rats by adenovirus-mediated gene transfer of brain-derived neurotrophic factor in vivo not only promotes nerve regeneration but also protects the neurons in the spinal cord.

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