A型肉毒毒素重链对大鼠脊髓损伤局部颈上神经节蛋白10的表达及神经突起再生的影响

关键词：肉毒毒素  A型肉毒毒素重链  脊髓损伤  颈上神经节蛋白10  神经突起  神经再生

Keywords:Botulinum neurotoxins  Botulinum neurotoxin serotype A heavy chain  spinal cord injury  superior cervical ganglion 10  neuronal processes  nerve regeneration

摘要：

      摘要 目的：探讨人工重组A型肉毒毒素重链（BoNT/A 重链）对大鼠脊髓损伤局部颈上神经节蛋白10（SCG10）表达、神经突起再生以及损伤侧后肢感觉及运动功能的影响，为阐明BoNT/A 重链促神经突起生长的作用提供实验依据。 方法：大鼠单侧腰段脊髓横断损伤模型基础上局部及鞘内给予BoNT/A 重链；于用药后不同时间提取局部蛋白进行双向电泳，并根据双向电泳提示的蛋白点群变化的分子量和等电点，选取SCG10为目标蛋白行Western Blot检测及神经突起测定；通过抓力和热痛敏实验测试损伤侧后肢的运动和感觉功能情况。 结果：①BoNT/A重链可干预脊髓损伤后局部蛋白谱的变化，MW位于18—25kDa、等电点在5—7范围的蛋白点可以作为蛋白点群变化的代表之一；②作为分子量位于18—25kDa/等电点在5—7的蛋白成分之一的SCG10在单纯损伤时较正常对照组其表达有所增高，给予BoNT/A 重链后, SCG10的表达显著增强（P<0.05）；③免疫荧光结果显示：单纯损伤时SCG10的阳性表达主要分布于损伤周边细胞的胞体，应用BoNT/A 重链后，SCG10阳性分布于胞浆和细胞突起且以损伤近头端的周边区域较为显著；④突起测定结果显示：BoNT/A 重链可促进脊髓损伤周边神经细胞突起增长，包括突起数目增多及含有突起的神经细胞百分比增多（P<0.05）；⑤给予BoNT/A重链后，损伤侧后肢肌力明显优于单纯损伤组（P<0.05），但感觉功能未见明显改善。 结论：脊髓损伤局部给予BoNT/A 重链可促进生长相关蛋白SCG10的表达并促进损伤周边神经细胞突起生长，改善肌肉抓力。BoNT/A重链有助于脊髓损伤后的再生修复。

Abstract:

      Abstract Objective: To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain (BoNT/A heavy chain) on the expression of superior cervical ganglion 10 (SCG10) and its influence in neurites spreading after unilateral lumbar spinal cord injury (SCI) in rats. Meanwhile, the ipsilateral thermal hyperalgesia and grasp power were also measured in order to evaluate the role of BoNT/A heavy chain in promoting spinal functional repair after SCI. Method: Recombinant BoNT/A heavy chain was locally and intrathecally administrated onto unilateral lumbar spinal cord injured rats. First of all, two-demension gel electrophoresis was proceeded for protein extracted from injured SCI tissues，SCG10 was chosen as representative of the altered proteins for morphological feature assessment. On the other hand, thermal hypergelsia and grasp power at ipsilateral hindlimb were also measured to test the sensory and motor function. Result: ①BoNT/A heavy chain may intervened the local expression pattern of protein after SCI. ②SCG10，representative of growth-related protein showed mild increase while SCI only (P<0.05) and remarkable increase after BoNT/A heavy chain application. ③The inmmuno-fluorescence on SCI sections exhibited that the SCG10 positive points mainly appeared at the cyton surrounding the injury. After BoNT/A heavy chain treatment, SCG10 positive points distributed among the cytoplasm and neuronal processes, especially the rostral area surrround the injury. ④The measurement of SCG10 positive neuronal process revealed that BoNT/A heavy chain promote the growth of neuronal process, including the process length and number percentage of cells with identifiable process (P<0.05).⑤The application of BoNT/A heavy chain after SCI didn’t affect the sensitive function, but significantly improved the ipsilateral hindlimb grasp power (P<0.05). Conclusion: The application of BoNT/A heavy chain after SCI was benefited on stimulating the expression of SCG10, a protein related to growth and regeneration. Also, BoNT/A heavy chain stimulated the outgrowth of neuronal process after SCI and facilitated the recovery of hind limb motor function after SCI.