神经干细胞及衍生支持因子在大鼠脊髓损伤后的变化

Changes in neural stem cells and their derived supporting factor in injured spinal cord tissues of rats

目的:神经干细胞及其支持因子均与中枢神经损伤后的神经再生有关.但目前有关神经干细胞及其衍生支持因子在体内和脊髓损伤后是否有变化的证据较少。观察神经干细胞及其衍生的神经干细胞支持因子在大鼠脊髓损伤后的变化规律.探讨它们之间的相互关系。方法:实验于2005-09/2006-03在南通大学神经科学研究所实验室完成,①实验材料:清洁级SD大鼠27只由南通大学实验动物中心提供,将12只大鼠分为假手术组和脊髓损伤组用于免疫组织化学.另15只用于RT-PCR检测.分为假手术组和脊髓损伤组.实验过程中对动物处置符台动物伦理学标准。②实验方法:按Allen重物打击法制备大鼠脊髓损伤模型,假手术组仪切除椎板,未行脊髓打击。用免疫组织化学方法鉴定脊髓组织中神经干细胞标志物nestin、胶质细胞标志物GFAP和神经干细胞衍生的神经干细胞支持因子阳性细胞在脊髓损伤后8.16 d的数量和形态变化;采用半定量RT-PCR检测神经干细胞衍生的神经干细胞支持因子mRNA在脊髓中的表达及损伤后4、8、12、16 d的变化。结果:①假手术组脊髓中央管附近出现巢蛋白阳性细胞脊髓损伤后8 d.巢蛋白阳性细胞增多,细胞变大.向外周迁移,并发出突起,以着力侧为著,胶质纤维酸性蛋白阳性细胞出现增多,并出现表达神经干细胞衍生的神经干细胞支持因子阳性类似神经元形态的细胞。脊髓损伤后16 d,nestin阳性细胞数量明显减少,以GFAP阳性细胞增生为主.神经干细胞衍生的神经干细胞支持因子阳性细胞少见。②RT-PCR检测神经干细胞支持因子mRNA在正常大鼠脊髓中有表达,脊髓损伤后4 d神经干细胞支持因子的mRNA表达上升.损伤8 d为高峰,16 d恢复到近正常水平。结论:①在正常大鼠脊髓中央管附近存在神经干细胞.在脊髓损伤后出现增殖,随后可能分化为神经胶质细胞。②在正常脊髓有神经干细胞支持因子表达.脊髓损伤后神经干细胞支持因子的mRNA表达随神经干细胞增殖和分化而增高和下降.两者在脊髓损伤修复中关系密切。

AIM： Neural stem cells （NSCs） and their supporting factors are related to nerve regeneration after central nerve injury, but there are few evidences for the changes of NSCs and their derived supporting factors. This study explores the change of NSCs and gene expression of stem cell-derived neural stem/progenitor cell supporting factor （SDNSF） in the injured spinal cord tissues of rats, and investigates the relation between the NSCs and expressions of SDNSF. METHODS： The experiment was carried out in the laboratory of Nantong University Neuroscience Institute between September 2005 and March 2006. ①A total of 27 SD rats, cleaning grade, were offered by the Animal Experimental Center of Nantong University. Twelve rats used for immunohistochemistry assay were divided into sham operation group and spinal cord injury group, while other 15 rats used for RT-PCR detection were assigned into sham operation group and spinal cord injury group. All the disposals were in agreement with the ethical standard for animals.②The spinal cord contusion model of rats was established according to Allen＇s falling strike method. Laminectomy was conducted without strike in the sham operation group. The quantity and morphology of NSCs, gila cells and SDNSF-positive cells were observed by immunochemistry to detect expressions of nestin, glial fibrillary acidic protein （GFAP） and SDNSF in the injured spinal cord on days 8 and 16 after injury. The expression of SDNSF mRNA in the injured spinal cord was studied by semi-quantitative RT-PCR on days 4, 8, 12, 16 after injury. RESULTS： ①There were nestin-positive cells about central canal of spinal cord in the sham operation group. The nestin-positive cells showed erupted roots, migrated peripherally and proliferation, some GFAP-positive cells emerged and the SDNSF-positive cells were observed, which were similar to neuron on morphology on day 8 after injury. The quantity of nestin-positive cells decreased obviously, there were a number of GFAP-positive cells and few SDNSF-positive cells were showed on day 16 after injury. ②RT-PCR revealed that SDNSF mRNA expressed in normal rate, and the expression was up-regulated on day 4 after injury, peaked on day 8, and decreased to the normal level on day 16. CONCLUSION： ①There are some NSCs about the central canal of spinal cord, which proliferate and soon may differentiate to gila cell after injury in normal rats. ②SDNSF is expressed in the normal spinal cord. The expression of SDNSF mRNA in the spinal cord varies with injured time, accompanying the proliferation and differentiation of NSCs. There are intimate relations between NSCs and expression of SDNSF in the repair of spinal cord injury.