NT-3壳聚糖支架诱导成年大鼠创伤性脑损伤后海马神经网络形成

发布时间：2018-04-04 05:58

本文选题：创伤性脑损伤　切入点：NT-3壳聚糖支架　出处：《首都医科大学》2014年硕士论文

【摘要】：研究背景：创伤性脑损伤后，，导致神经元死亡或丢失，造成多种神经功能缺陷，其中海马损伤导致认知功能缺陷严重影响了患者的空间学习与记忆功能。传统的观点认为中枢神经通路是固定的、不能改变的。中枢神经死亡后不能再生，由胶质细胞替代丢失的神经细胞。近年研究表明在缺血损伤等条件下，成年大鼠脑海马内存在的内源性神经前体细胞可以不断增殖产生新生神经元。这为修复中枢神经损伤提供了理论基础。研究目的：创伤性脑损伤后移植入NT-3壳聚糖支架材料，研究损伤脑组织再生及损伤区边缘瘢痕情况；观察损伤区神经再生过程以及损伤区内新生神经元突触形成，并参与脑神经环路重建的研究。实验方法：本研究采用生物吸除装置造成大鼠脑海马CA1及以上皮层机械性损伤做为创伤性脑损伤的实验模型。实验动物随机分为三组：分别是单纯损伤组、单纯壳聚糖支架组、NT-3壳聚糖支架组。分别于术后3d、7d、14d、28d和60d，各实验组取4只动物灌杀取脑，冰冻切片免疫化学组织染色观察。NF染色观察损伤区内再生的神经元及神经纤维的情况；GFAP染色观察损伤边缘胶质瘢痕情况。Nestin免疫组织化学染色观察损伤区内神经前体细胞增殖情况；βtubulinⅢ免疫组织化学染色观察损伤区内分化的不成熟神经元；MAP2免疫组织化学染色观察损伤区内分化成熟的神经元。术后30d、60d灌杀动物，利用免疫电镜技术和免疫组织化学染色观察损伤区内再生神经元的突触超微结构。术后60d，应用神经示踪方法将BDA-FITC注射到损伤对侧CA3区，荧光显微镜下观察示踪剂走形情况。研究结果：免疫组织化学结果显示：创伤性脑损伤后60天，各组损伤区内都可见不同数量神经再生，统计数值后发现：NT-3壳聚糖支架组同单纯壳聚糖组、单纯损伤组存在明显的统计学差异；同时观察损伤边缘的胶质瘢痕情况发现NT-3壳聚糖支架组GFAP阳性细胞数明显少于单纯损伤组和单纯壳聚糖支架组。以上结果说明创伤性脑损伤后，移植NT-3壳聚糖支架可以促进损伤区内神经再生，抑制损伤区边缘胶质瘢痕的形成。进一步研究移植NT-3壳聚糖支架到损伤区后，观察神经前体细胞增殖分化为成熟神经元的过程。研究发现移植NT-3壳聚糖支架组，术后3天时nestin阳性细胞被激活，7天时nestin阳性细胞数达高峰，到14天出现下降趋势。观察发现脑损伤后14天βtubulinⅢ阳性细胞数增多，28天后阳性细胞数下降。研究发现脑损伤后28天MAP2阳性细胞数逐渐增多，此增长趋势延续到脑损伤后60天。统计数值发现：NT-3壳聚糖支架组nestin、βtubulinⅢ和MAP2阳性细胞数同其他对照组存在明显的统计学差异。移植NT-3壳聚糖支架组损伤后30天，通过免疫电镜方法观察损伤区内出现BrdU+MAP2+双阳性的新生神经元，并形成成熟突触结构。荧光显微镜下观察到BDA+神经细胞通过右侧海马CA3锥体细胞的轴突沿着海马联合到达左侧CA1区，在损伤区内观察到BDA+细胞，并且部分BDA+细胞表达MAP2+。研究结论：NT-3壳聚糖支架可以促进损伤区内神经再生，抑制损伤周边胶质瘢痕的形成。损伤区内神经前体细胞能够增殖分化为成熟神经元并形成神经突触，参与脑神经网络的修复重建。
[Abstract]:Background : After traumatic brain injury , the death or loss of neurons leads to a variety of neurological deficits , in which the damage of the hippocampus leads to a severe impairment of cognitive function . The traditional view suggests that the central nervous pathway is fixed and cannot be changed . In recent years , the endogenous neural progenitor cells present in the hippocampus of adult rats can proliferate and produce new neurons . This is a theoretical basis for repairing the central nervous system injury .

Objective : To study the regeneration of injured brain tissue and the scar of injured area after traumatic brain injury .
Observe the nerve regeneration process in the injured area and the synaptic formation of neonatal neurons in the injured area , and participate in the study of the reconstruction of the brain nerve loop .

The experimental animal model of traumatic brain injury was studied by means of biosorption device . The experimental animals were randomly divided into three groups : simple injury group , chitosan stent group and NT - 3 chitosan stent group . The experimental animals were divided into three groups : simple injury group , simple chitosan stent group and NT - 3 chitosan stent group .
GFAP staining was used to observe the glial scar in the injured area . Nestin immunohistochemistry staining was used to observe the proliferation of neural progenitor cells in the injured area .
Non - mature neurons in the injured area were observed by immunohistochemical staining of 尾 - 鈪⑩參 .
The synaptic ultrastructure of regenerated neurons in the injured area was observed by immunohistochemical staining of MAP2 . After the operation , the synaptic ultrastructure of the regenerated neurons in the injured area was observed by immunoelectron microscopy and immunohistochemical staining . After operation 60 days after operation , the experimental tracer was injected into the injured contralateral CA area under the fluorescence microscope .

The results showed that after traumatic brain injury 60 days after traumatic brain injury , different numbers of nerve regeneration were observed in each group , and it was found that NT - 3 chitosan stent group had obvious statistical difference compared with simple chitosan group and simple injury group .
It was found that the number of GFAP - positive cells in NT - 3 chitosan stent group was significantly lower than that of simple injury group and simple chitosan stent group .

Conclusion : NT - 3 chitosan scaffold can promote nerve regeneration in the injured area and inhibit the formation of glial scar in the injured peripheral area . The neural progenitor cells in the injured area can proliferate into mature neurons and form neuroses , and participate in the repair and reconstruction of cranial nerve network .

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R651.15

【参考文献】