A型肉毒素重链干预对大鼠脊髓损伤后生长相关蛋白表达的影响

目的探讨人工重组A型肉毒素重链(Bo NT/A重链)对在体大鼠脊髓损伤局部生长相关蛋白表达的影响,为阐明Bo NT/A重链促神经突起再生机制提供依据。方法建立大鼠单侧腰段脊髓横断损伤模型并局部及鞘内给予Bo NT/A重链;对用药后不同时间局部骨髓组织进行双向电泳检测总体蛋白表达;选择生长相关蛋白-43(GAP-43)和颈上神经节蛋白10(SCG 10)进行蛋白印迹和免疫荧光检测以观察二者在Bo NT/A重链影响下的表达及分布。结果 (1)单侧腰髓离断损伤模型动物呈现明显单侧运动及感觉功能障碍;(2)脊髓损伤后给予Bo NT/A重链可影响损伤局部蛋白表达谱变化:Bo NT/A重链可引起损伤局部蛋白点群在变化的基础上逆转或表达进一步增加,尤以MW位于35~45 k Da及18~25 k Da、等电点在5~7范围的蛋白点表现明显;(3)蛋白印迹和免疫荧光染色结果提示:Bo NT/A重链可促进p-GAP 43和SCG 10的表达(P0.05),且p-GAP 43及SCG 10主要以损伤周围细胞阳性明显,胞浆及突起皆呈阳性。结论脊髓损伤后给予Bo NT/A重链可促进脊髓损伤后选择性生长相关蛋白p-GAP 43和SCG 10的表达。

Effect of botulinum neurotoxin type A heavy chain on the growth-related proteins after spinal cord injury in rats

Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain (BoNT/ A heavy chain) on local proteins which are related to nerve growth after spinal cord injury in rats, and to get some experimental evidence to explain the mechanism of BoNT/ A heavy chain in stimulating neuritogenesis. Methods Recombinant botulinum neurotoxin serotype A heavy chain was applied locally or intrathecally to rats with ipsilateral semidissociated lumbar spinal injury. Local spinal tissue was extracted for general protein expression by two dimension electrophoresis plus nitrate silver staining after different time period of injury. Based on the results of 2-D gelelectrophoresis, growth-associated protein 43 (GAP-43) and of superior cervical ganglion 10 (SCG 10) were selected to examine the changes of their expression and distribution features under BoNT/ A heavy chain administration using SDS-PAGE, western blot and immunofluorescence. Results (1) The model of spinal cord injury (SCI) in this study was an ipsilateral semi?dissociated lumbar SCI in rat. The rats showed obvious motor and sensory dysfunction in the ipsilateral hind limb. (2) The results from 2-D gel electrophoresis plus nitrate silver staining showed that the administration of BoNT/ A heavy chain based on SCI altered the local protein expression pattern. The decrease or increase in the expression of some protein dots / dots group was clearly seen after single SCI. However, these changes were transformed by BoNT/ A heavy chain treatment, which appeared as a reversed pattern turning toward that in control group or further increased expression upon SCI, such as the dots located respectively at 35 -45 kDa and 18 -25 kDa level, pI between 5 -7. In addition, the expression of the two dots located at the level as above increased after SCI only, and showed further increase in their expression with BoNT/ A heavy chain intervention. (3) The changes of selective GAP-43 and SCG 10 expression and distribution by western blot and immunofluorescence indicated that the administration of BONT/ A heavy chain based on SCI amplified the expression of GAP-43 and SCG 10 (P < 0.05). Meanwhile, the positive immuonfluorescent staining for both GAP-43 and SCG 10 mainly distributed nearby the proximal area of injury, both cytoplasm and neuronal processes were positively stained. Conclusions Intrathecal delivery of BoNT/ A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.