【作者】 李芳；

【导师】 罗学港；

【作者基本信息】 中南大学 ， 神经生物学， 2008， 博士

【摘要】 第一部分条件性损伤在脊髓损伤后再生中的作用目的:利用示踪技术,观察条件性损伤(坐骨神经损伤或者腰5运动根损伤)对脊髓背索损伤后薄束、楔束再生的影响。方法:成年健康雌性SD大鼠,随机分为正常组、单纯脊髓损伤组(脊髓背索损伤)和合并损伤组(条件性损伤1周后行脊髓背索损伤)。脊髓背索损伤后,动物存活2周。在脊髓背索损伤同时,于脊髓损伤区头侧端5mm处注射快蓝(Fast blue,FB)逆行示踪背根节感觉神经元,动物处死前3天于坐骨神经注射霍乱毒素亚单位B(Cholera Toxin B Subunit,CTB)顺行示踪脊髓上行感觉束,观察脊髓损伤后薄束、楔束的再生情况。结果:正常大鼠薄束核和楔束核可见CTB阳性纤维;单纯脊髓损伤组薄束核和楔束核则未发现CTB阳性纤维,CTB阳性纤维终止于脊髓损伤区尾侧端,形成膨大样结构,没有进入胶质瘢痕;合并损伤组,可见CTB标记的上行感觉束纤维,穿过脊髓损伤区进入损伤区头侧端,胶质瘢痕中CTB阳性纤维数目较单纯脊髓损伤组多。FB逆行示踪显示,合并损伤组背根节中FB标记的感觉神经元数目较单纯脊髓损伤组多。结论:条件性损伤能促进脊髓背索损伤后薄束、楔束的再生。第二部分条件性损伤促进脊髓损伤后再生的作用机制目的:观察条件性损伤后脊髓和背根节中脑源性神经营养因子(Brain-derived neurotrophin factor,BDNF)的表达情况及中和了内源性BDNF后条件性损伤对脊髓再生的影响,探讨条件性损伤在脊髓损伤后再生中的作用机制。方法:成年健康雌性SD大鼠,随机分为正常组、单纯条件性损伤组、单纯脊髓损伤组和合并损伤组,合并损伤组动物腹腔给予BDNF抗血清或正常羊血清(NSS),术后动物存活1天、7天或14天。采用免疫组织化学、原位杂交和酶联免疫吸附实验(ELISA)方法检测BDNF在损伤脊髓局部、脊髓腰骶膨大部及双侧L4、L5背根节中的表达,并结合FB逆行示踪观察FB阳性神经元BDNF的表达情况。用FB逆行示踪背根节感觉神经元和CTB顺行标记上行感觉束,观察BDNF抗血清处理后脊髓再生的情况。条件性损伤1周后,取背根节神经元和背根节组织进行体外培养,通过βⅢ-tubulin免疫荧光染色观察BDNF抗血清处理后背根节神经元突起的生长情况。结果:单纯条件性损伤组,损伤侧的脊髓腰骶膨大部、背根节中BDNF蛋白及mRNA表达均上调。与单纯脊髓损伤组相比,合并损伤组在脊髓损伤的尾侧端可见较多的BDNF阳性纤维,这些纤维呈膨体样结构,主要分布于损伤脊髓尾侧端的白质里,合并损伤组FB标记的感觉神经元数目也较单纯脊髓损伤组多,且大部分FB标记的感觉神经元是BDNF阳性的神经元。中和内源性BDNF后,合并损伤组脊髓CTB标记的纤维终止于脊髓空洞前,仅有极少数纤维长入损伤区。通过测量由损伤区尾侧端长入损伤区的再生轴突长度发现,BDNF抗血清处理组再生轴突的长度明显短于NSS处理组,同时,背根节中FB标记的感觉神经元数目也显著少于NSS处理组。背根节神经元体外培养和Matrigel里培养背根节组织显示,BDNF抗血清处理后,背根节神经元和背根节组织神经突起的长度较NSS组明显缩短。结论:条件性损伤促进脊髓损伤后的轴突再生机制可能与脊髓和背根节中BDNF的表达增加有关。第三部分BDNF参与条件性损伤促进脊髓损伤再生作用的可能机制目的:通过观察条件性损伤或合并损伤结合BDNF抗血清处理后,脊髓和背根节中生长相关蛋白-43(growth associated protein-43,GAP-43)、磷酸化细胞外信号调节激酶(phosphorylated extracellularsignal-regulated kinase,p-Erk)和RhoA的表达变化,探讨BDNF参与条件性损伤促进脊髓损伤再生作用的可能机制。方法:成年健康雌性SD大鼠,随机分为正常组、单纯条件性损伤组、单纯脊髓损伤组和合并损伤组,单纯条件性损伤组和合并损伤组动物术后给予BDNF抗血清或NSS处理,大鼠存活3天、7天或14天。用免疫组织化学方法、谷胱甘肽转移酶pull down(GST-pulldown)及蛋白质免疫印迹等,检测单纯条件性损伤组背根节中GAP-43和p-Erk的表达,单纯脊髓损伤组和合并损伤组脊髓损伤局部GAP-43和RhoA的表达情况,及BDNF抗血清处理后,上述指标的表达变化情况。结果:与正常对照相比,单纯条件性损伤后1周,背根节中GAP-43和p-Erk阳性的感觉神经元数目显著上调,BDNF抗血清处理后,GAP-43阳性和p-Erk阳性的感觉神经元比例下调。脊髓损伤后3天,单纯脊髓损伤组GAP-43和RhoA表达上调,7天达到高峰,并持续到损伤后14天。合并损伤组GAP-43的表达明显上调,较单纯脊髓损伤组高,但RhoA表达下调。BDNF抗血清处理后,合并损伤组脊髓损伤局部GAP-43的表达较NSS组下调,RhoA表达上调。结论:在条件性损伤促进脊髓损伤后再生过程中,BDNF可能通过上调GAP-43的表达,激活p-Erk信号通路及下调RhoA发挥作用。更多还原

【Abstract】 PartⅠThe role of preconditioning lesion in the regeneration of injured spinal cordObjective:To observe the effect of preconditioning lesion（sciatic nerve transection or L5 ventral root transection（L5VRT）one week prior to dorsal column injury）on the regeneration of fasciculus gracilis and fasciculus cuneams in the injured spinal cord.Methods:Adult female Sprague-Dawley（SD）rats were randomly divided into normal group,spinal cord injury group and combined injury （preconditioning lesion combined with spinal cord injury）group.The rats survived for 2 weeks after spinal cord injury.FB was injected immediately after spinal cord injury into 5mm rostra1 to the spinal cord lesion site to retrogradely label sensory neurons in the dorsal root ganglia （DRG）.CTB was injected into the sciatic nerve to anterogradely label ascending axons 3 days before sacrifice.Results:In the normal spinal cord,CTB-ir was observed in the gracile and cuneatus nucleus.After spinal cord injury,no CTB labeled nerve terminals were observed in the gracile and cuneatus nucleus,axons were stopped before the cavity and no axons were found in the glia scar of injured spinal cord.In the combined injury group,the CTB-ir axons were found caudal to,within and rostral to the cavity of injured spinal cord.Moreover,the number of CTB-labeled axons in the glia scar in the combined injury group was greater than those in the spinal cord injury group.In the ipsilateral DRG,the number of FB-labeled sensory neurons was significantly increased in the combined injury group compared to the spinal cord injury group.Conclusion:Preconditioning lesion promotes regeneration of ascending sensory tract in the injured spinal cord. PartⅡThe mechanism of preconditioning lesion on the enhanced regeneration of injured spinal cordObjective:To investigate the mechanism of preconditioning lesion on the enhanced regeneration of injured spinal cord,the expression of brain-derived neurotrophic factor（BDNF）was observed in spinal cord and DRG and the regeneration of the injured spinal cord was evaluated after preconditioning lesion with BDNF antiserum treatment.Methods:Adult female Sprague-Dawley（SD）rats were randomly divided into normal group,preconditioning lesion group,spinal cord injury group and combined injury group.Rats in the combined injury group were intraperitoneally injected with BDNF antiserum or normal sheep serum（NSS）.Animals were allowed to survive for 1,7 and 14 days. Immunohistochemistry,in situ hybridization and ELISA were used to detect the expression of BDNF in the injured spinal cord,lumbar spinal cord and bilateral lumbar 4,5 DRG.And also,BDNF expression was detected in FB labeled neurons of the DRG.The regeneration was evaluated with FB and CTB labeling in the combined injury group after BDNF neutralization.βⅢ-tubulin immunohistochemistry was carried out to evaluate the neurite growth of the cultured DRG neurons and explants after BDNF neutralization.Results:It was found that the expression of BDNF protein and mRNA in the DRG,lumbar spinal cord and injured spinal cord were up-regulated in the preconditioning lesion group.Compared with the spinal cord injury group,more BDNF positive fibers with a club-like shape in the white matter were seen in the spinal cord segment caudal to lesion site ipsilateral to preconditioning lesion side in the combined injury group.Besides,in the ipsilateral DRG,there were more FB+ neurons, most of which were immunoreactive for BDNF,in the combined injury group than in the spinal cord injury group.After treated with BDNF antiserum,axons were stopped before the cavity and only very few axons grew short distance into the lesion site in the combined injury group.The length of axons from the caudal boundary of the lesion site towards the lesion site in the NSS group was longer than that of BDNF antiserum treated rats.In the ipsilateral DRG,the number of FB-labeled regenerating neurons was significantly reduced in the BDNF antiserum treated group,compared to those in the NSS group.Neurite length of cultured DRG neurons or explants in the Matrigel was significantly shorter in the BDNF antiserum treated group than that in the NSS group.Conclusion:The up-regulation of BDNF maybe involved in the enhanced regeneration of injured spinal cord induced by preconditioning lesion. PartⅢThe mechanism of endogenous BDNF in the enhanced regeneration of injured spinal cord after preconditioning lesionObjective:To study the mechanism of endogenous BDNF in the enhanced regeneration of injured spinal cord after preconditioning lesion by observing the expression of GAP-43,p-Erk and RhoA with or without neutralizing endogenous BDNF.Methods:Adult female Sprague-Dawley（SD）rats were randomly divided into preconditioning lesion group,spinal cord injury group and combined injury group.Rats in the preconditioning lesion group and combined injury group were intraperitoneally injected with BDNF antiserum or normal sheep serum（NSS）.Immunohistochemistry, immunoblotting or RhoA pull-down assays were performed to study the expression of GAP-43 and p-Erk or the activation of RhoA in the different groups with or without neutralizing endogenous BDNF.Results:One week after preconditioning lesion in the NSS treated animals,the number of GAP-43+ and p-Erk+ neurons was significantly increased in the ipsilateral DRG.The number of GAP-43+ and p-Erk+ neurons in the ipsilateral DRG was reduced after BDNF antiserum treatment.The expression of GAP-43 and RhoA was upregulated at the third days after spinal cord injury and maintained until the 14^thday.The highest level of GAP-43 and RhoA expression presented at the 7^thdays after spinal cord injury.After spinal cord injury,the level of GAP-43 was higher but RhoA was lower in the combined injury group than that of the spinal cord injury group.After treatment with the BDNF antiserum,the expression of GAP-43 was reduced and RhoA was upregulated after BDNF antiserum treatment in the combined injury group.Conclusion:The upregulation of GAP-43,activation of p-Erk signal pathway and downregulation of RhoA by increased BDNF maybe involved in the enhanced regeneration of injured spinal cord after preconditioning lesion.更多还原