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
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摘要
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(P<0.05),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。
Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain( Bo NT/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 Bo NT/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 gel electrophoresis,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 Bo NT/A heavy chain administration using SDSPAGE,western blot and immunofluorescence. Results( 1) The model of spinal cord injury( SCI) in this study was anipsilateral 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 BoN T/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 BoN T/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 kD a and 18-25 kD a 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 BoN T/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 BoN T/A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.
Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain( Bo NT/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 Bo NT/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 gel electrophoresis,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 Bo NT/A heavy chain administration using SDSPAGE,western blot and immunofluorescence. Results( 1) The model of spinal cord injury( SCI) in this study was anipsilateral 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 BoN T/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 BoN T/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 kD a and 18-25 kD a 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 BoN T/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 BoN T/A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.
引文
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[18]Fernandes KJ,Fan DP,Tsui BJ,et al.Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons:differential regulation of GAP-43,tubulins,and neurofilamentM[J].J Comp Neurol,1999,414(4):495-510.
[19]Fagoe ND,van Heest J,Verhaagen J.Spinal cord injury and the neuron-intrinsic regeneration-associated gene program[J].Neuromolecular Med,2014,16(4):799-813.
[20]高美玲,王红,张彩云,等.血清型A肉毒杆菌神经毒素重链对Neuro-2a细胞的促神经突起再生作用[J].中国病理生理杂志,2015,31(12):2221-2227.
[21]王红,高美玲,兰婧,等.小鼠神经母细胞瘤细胞株用于A型肉毒毒素重链体外实验的可行性研究[J].中华细胞与干细胞杂志:电子版,2015,5(4):23-28.
[22]Ma TC,Willis DE.What makes a RAG regeneration associated?[J].Front Mol Neurosci,2015,8:43.
[23]Lieberman AR.The axon reaction:a review of the principal features of perikaryal responses to axon injury[J].Int Rev Neurobiol,1971,14:49-124.
[24]Grafstein B.The nerve cell body response to axotomy[J].Exp Neurol,1975,48(3 pt.2):32-51.
[25]Skene JH,Willard M.Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems[J].J Cell Biol,1981,89(1):96-103
[26]Skene JH.Axonal growth-associated proteins[J].Annu Rev Neurosci,1989,12:127-156.
[27]Ozon S,Maucuer A,Sobel A.The stathmin family—molecular and biological characterization of novel mammalian proteins expressed in the nervous system[J].Eur J Biochem,1997,248(3):794-806.
[28]Wang J,Shan C,Cao W,et al.SCG 10 promotes non-amyloidogenic processing of amyloid precursor protein by facilitating its trafficking to the cell surface[J].Hum Mol Genet,2013,22(24):4888-4900.
[29]Shin JE,Geisler S,Diantonio A.Dynamic regulation of SCG 10in regenerating axons after injury[J].Exp Neurol,2014,252(3):1-11.
[2]Giger RJ,Hollis ER,Tuszynski MH.Guidance molecules in axon regeneration[J].Cold Spring Harb Perspect Biol,2010,2(7):a001867.
[3]Simpson LA,Eng JJ,Hsieh JT,et al.The health and life priorities of individuals with spinal cord injury:A systematic review[J].J Neurotrauma,2012,29(8):1548-1555.
[4]Ferguson TA,Son YJ.Extrinsic and intrinsic determinants of nerve regeneration[J].J Tissue Eng,2011,2(1):2041731411418392.
[5]He Z.Intrinsic control of axon regeneration[J].J Biomed Res,2010,24(1):2-5.
[6]Burgen AS,Dickens F,Zatman LJ.The action of botulinum toxin on the neuro-muscular junction[J].J Physiol,1949,109(1-2):10-24.
[7]Schiavo G,Rossetto O,Benfenati F,et al.Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus[J].Ann N Y Acad Sci,1994,710:65-75.
[8]Navarrete AL,Rafferty KL,Liu ZJ,et al.Botulinum neurotoxin type A in the masseter muscle:effects on incisor eruption in rabbits[J].Am J Orthod Dentofacial Orthop,2013,143(4):499-506.
[9]Park SY,Park YW,Ji YJ,et al.Effects of a botulinum toxin type A injection on the masseter muscle:An animal model study[J].Maxillofac Plast Reconstr Surg,2015,37(1):10.
[10]Ansved T,Odergren T,Borg K.Muscle fiber atrophy in leg muscles after botulinum toxin type A treatment of cervical dystonia[J].Neurol,1997,48(5):1440-1442.
[11]Durham PL,Cady R,Cady R.Regulation of calcitonin gene-related peptide secretion from trigeminal nerve cells by botulinum toxin type A:implications for migraine therapy[J].Headache,2004,44(1):35-42.
[12]Schiavo G,Matteoli M,Montecucco C.Neurotoxins affecting neuroexocytosis[J].Physiol Rev,2000,80(2):717-766.
[13]Lacy DB,Tepp W,Cohen AC,et al.Crystal structure of botulinum neurotoxin type A and implications for toxicity[J].Nat Struct Biol,1998,5(10):898-902.
[14]李夏青.肉毒杆菌毒素的临床应用及其前景[M].北京:知识产权出版社,2012:1-200.
[15]Ayyar BV,Tajhya RB,Beeton C,et al.Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin[J].Sci Rep,2015,5:15776.
[16]邓亚南,刘艳芳,陈建平,等.经大鼠腰骶部鞘内置管给药技术的研究[J].中国医学创新,2014,11(18):33-35.
[17]Goldberg JL,Klassen MP,Hua Y,et al.Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells[J].Science,2002,296(5574):1860-1864.
[18]Fernandes KJ,Fan DP,Tsui BJ,et al.Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons:differential regulation of GAP-43,tubulins,and neurofilamentM[J].J Comp Neurol,1999,414(4):495-510.
[19]Fagoe ND,van Heest J,Verhaagen J.Spinal cord injury and the neuron-intrinsic regeneration-associated gene program[J].Neuromolecular Med,2014,16(4):799-813.
[20]高美玲,王红,张彩云,等.血清型A肉毒杆菌神经毒素重链对Neuro-2a细胞的促神经突起再生作用[J].中国病理生理杂志,2015,31(12):2221-2227.
[21]王红,高美玲,兰婧,等.小鼠神经母细胞瘤细胞株用于A型肉毒毒素重链体外实验的可行性研究[J].中华细胞与干细胞杂志:电子版,2015,5(4):23-28.
[22]Ma TC,Willis DE.What makes a RAG regeneration associated?[J].Front Mol Neurosci,2015,8:43.
[23]Lieberman AR.The axon reaction:a review of the principal features of perikaryal responses to axon injury[J].Int Rev Neurobiol,1971,14:49-124.
[24]Grafstein B.The nerve cell body response to axotomy[J].Exp Neurol,1975,48(3 pt.2):32-51.
[25]Skene JH,Willard M.Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems[J].J Cell Biol,1981,89(1):96-103
[26]Skene JH.Axonal growth-associated proteins[J].Annu Rev Neurosci,1989,12:127-156.
[27]Ozon S,Maucuer A,Sobel A.The stathmin family—molecular and biological characterization of novel mammalian proteins expressed in the nervous system[J].Eur J Biochem,1997,248(3):794-806.
[28]Wang J,Shan C,Cao W,et al.SCG 10 promotes non-amyloidogenic processing of amyloid precursor protein by facilitating its trafficking to the cell surface[J].Hum Mol Genet,2013,22(24):4888-4900.
[29]Shin JE,Geisler S,Diantonio A.Dynamic regulation of SCG 10in regenerating axons after injury[J].Exp Neurol,2014,252(3):1-11.