NT3-壳聚糖活性材料支架诱导内源性神经发生修复成年大鼠脑损伤的研究

发布时间：2018-01-21 23:39

本文关键词： 创伤性脑损伤 NT3-壳聚糖神经发生内源性神经干细胞神经网络功能恢复微环境　出处：《首都医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:明确NT3-壳聚糖载体能否促进创伤性脑损伤(traumatic brain injury,TBI)后损伤区内新生神经元的产生,并探讨这些新生神经元能否功能性整合入脑环路以修复脑功能;观察TBI及NT3-壳聚糖对内源性神经干细胞(neural stem cells,NSCs)的激活情况,阐明NT3-壳聚糖对海马齿状回(dental gyrus,DG)颗粒细胞下层(subgranular zone,SGZ)和侧脑室室旁区后部(posterior periventricle,pPV)NSCs的增殖、迁移和神经向分化的影响;探讨NT3-壳聚糖能否改善损伤区的微环境。方法:应用自制的脑吸除装置制备成年大鼠吸除性脑损伤模型,损伤范围为海马CA1区及以上皮质,术后立即向损伤区植入含或不含有神经营养因子3(neurotrophin 3,NT3)的壳聚糖载体,实验分为单纯壳聚糖组和NT3-壳聚糖载体组,同时设假手术组(只打开骨窗,不接受TBI手术)和损伤对照组(接受TBI手术,但术后不给予任何治疗措施)。HE染色观察各组脑组织再生情况;大鼠术后立即腹腔注射BrdU以标记增殖的细胞,BrdU/Tuj1和BrdU/NeuN染色检测术后不同时间点损伤区新生神经元的再生,同时应用vGluT1和GAD67染色观察损伤区内不同类型神经元的再生;BrdU/Tuj1/synapsin1和PSD95免疫荧光标记及MAP2免疫电镜检测损伤区内新生神经元之间的突触形成;MED64平面微阵列记录系统检测损伤区内新生神经元之间及新生神经元与宿主脑之间的功能性神经网络形成;BDA神经示踪观察再生神经元在海马内的神经环路重建;morris水迷宫评价大鼠的空间学习记忆功能。为了检测不同脑区nscs的增殖,大鼠灌流前48小时开始腹腔注射brdu,于术后不同时间点观察并计数海马dg不同亚区和ppv区增殖细胞的数量;brdu/neun/gfap免疫荧光三标记检测dg增殖细胞的命运;brdu/dcx免疫荧光染色及dil体内标记观察sgz和ppv增殖nscs的迁移。cd45和cd11b染色观察nt3-壳聚糖对tbi后巨噬细胞和小胶质细胞的影响,实时定量pcr检测脑内促炎和抗炎因子的表达水平,reca-1染色观察损伤区内的血管再生。结果:1.brdu/tuj1和brdu/gfap/neun染色结果显示,nt3-壳聚糖能够促进损伤区内成熟神经元的再生,同时抑制胶质反应,并且新生神经元包含谷氨酸能神经元和gaba能神经元。2.brdu/tuj1/synapsin1和psd95免疫荧光染色及map2免疫电镜结果显示,nt3-壳聚糖促进损伤区内新生神经元之间的突触形成。med64进一步证明,nt3-壳聚糖促进新生神经元之间及新生神经元和宿主脑之间功能性神经网络的建立。bda神经示踪结果显示,新生神经元可重建受损的海马环路。3.morris水迷宫结果表明,nt3-壳聚糖可以改善损伤后期大鼠的认知功能,同时恢复海马内schaffer侧支-ca1突触的ltp特性。4.brdu/nestin染色结果显示,nt3-壳聚糖可激活内源性nscs,并促进它们向损伤区的迁移。5.brdu染色结果显示,tbi增加损伤同侧dgbrdu+细胞数,nt3-壳聚糖进一步增加tbi所致的增殖反应,术后各时间点损伤同侧nt3-壳聚糖组的增殖细胞均多于单损组。对dg增殖细胞的分布分析结果显示,术后不同时间点nt3-壳聚糖组和单损组brdu+细胞大部分仍位于神经发生区sgz区,且nt3-壳聚糖组阳性细胞数多于单损组,其余细胞分布于dg非神经发生区即分子层和门区。6.brdu/dcx染色结果显示,nt3-壳聚糖增加sgzbrdu+/dcx+细胞数,同时增加BrdU+/Dcx+细胞的比例,说明NT3-壳聚糖不仅增加SGZ新生成神经细胞的数量,而且增加成神经细胞在增殖细胞中的比例。BrdU/NeuN/GFAP免疫标记显示,NT3-壳聚糖促进DG增殖细胞的存活,同时增加DG新生颗粒神经元的数量,促进DG神经发生。7.BrdU/Dcx染色结果表明,NT3-壳聚糖促进SGZ NSCs向损伤区的迁移。8.BrdU染色结果显示,NT3-壳聚糖增加TBI所致的损伤同侧pPV NSCs的增殖,BrdU/Dcx免疫染色和Dil体内标记实验显示,NT3-壳聚糖促进室周区NSCs向损伤区的迁移,损伤区内检测到的Dil+细胞部分表达NSCs标记物nestin,部分表达成神经细胞标记物Dcx,说明室周区NSCs可能参与了损伤区新生神经元的再生。9.CD45和CD11b染色结果显示,NT3-壳聚糖抑制损伤区内巨噬细胞的浸润和小胶质细胞的激活,同时恢复损伤周围脑组织不同状态小胶质细胞的比例。实时定量PCR结果显示,NT3-壳聚糖抑制TBI后促炎因子IL-1β和TNF-α的表达,同时增加抗炎因子IL-4和IL-10的水平,提示NT3-壳聚糖的抗炎作用。10.Reca-1荧光染色结果提示,NT3-壳聚糖促进损伤区内的血管再生,增加损伤区再生血管的密度和直径。结论:NT3-壳聚糖载体通过持续释放NT3神经营养因子,显著激活SGZ和pPV内源性NSCs,并促进它们向损伤区的异位迁移。同时该生物活性材料为损伤区提供了一个抗炎和促血管再生的微环境,有利于迁移至损伤区的NSCs分化为成熟的神经元,并建立功能性神经网络,进而修复受损的脑功能。这些结果为NT3-壳聚糖活性生物材料修复成年脑损伤的临床应用提供了理论基础和实验依据。
[Abstract]:Objective: to determine the NT3- of chitosan can promote traumatic brain injury (traumatic brain, injury, TBI) after injury of newborn neurons in the region, and to explore these new neurons can integrate into the brain functional loop to repair brain function; to observe the TBI and NT3- of Chitosan on endogenous neural stem cells (neural stem cells, NSCs the activation of NT3-, clarify) chitosan on hippocampal dentate gyrus (dental, gyrus, DG (subgranular) subgranular zone zone, SGZ) and periventricle rear (posterior periventricle, pPV NSCs) proliferation, migration and neural differentiation effect; explore the micro environment of NT3- chitosan can improve the damage zone methods: Using Self-made brain suction device for the preparation of adult rat model of brain injury aspiration, damage range for the CA1 region of the hippocampus and cortex above, immediately after surgery to implant damage zone containing or not containing neurotrophic factor 3 (Neurot Rophin 3, NT3) chitosan carrier, the experiment was divided into chitosan groups and NT3- chitosan group, sham operation group (only open bone window, do not accept the TBI surgery) and injury group (received TBI surgery, but postoperative given no treatment measures) were observed by.HE staining of brain tissue regeneration; rat operation immediately after intraperitoneal injection of BrdU labeled with the proliferation of cells, BrdU/Tuj1 and BrdU/NeuN were detected at different time points after new injury neuron regeneration, while using vGluT1 and GAD67 staining to observe the different types of neurons in the damage zone; formed between BrdU/Tuj1/synapsin1 and PSD95 by immunofluorescence and immunoelectron microscopy MAP2 detection of damage zone of newborn neurons synapse; functional neural network between MED64 plane microarray system measured in the damage zone of new neurons and neurons and host brain formation; BDA Neural tracing observation neural circuits in the hippocampus neuron regeneration and reconstruction; learning and memory function of Morris water maze in rats space. In order to detect NSCs in different brain regions of the proliferation of rats were perfused 48 hours before intraperitoneal injection of BrdU at different time points after operation to observe and count the sea horse DG different number of sub regions and PPV proliferation; brdu/neun/gfap immunofluorescence three markers DG proliferation cell fate; to observe the effect of Chitosan on nt3- TBI macrophages and microglia brdu/dcx immunofluorescence staining and DIL in SGZ and PPV marker to observe the proliferation of NSCs migration of.Cd45 and CD11b staining, the expression of real time quantitative PCR detection of brain proinflammatory and anti-inflammatory the observation of angiogenesis factor, damage zone. Results: 1.brdu/tuj1 reca-1 staining and brdu/gfap/neun staining showed that nt3- chitosan can promote the maturation of neurons and damage zone At the same time, inhibition of glial cells, and neurons contains glutamatergic neurons and GABA neurons.2.brdu/tuj1/synapsin1 and PSD95 immunofluorescence staining and MAP2 immunoelectron microscopy showed that nt3- chitosan to promote in the damage zone of new neurons synapse formation.Med64 further proved that nt3- chitosan to promote new neurons and between neurons and host brain function a neural network.Bda neural tracing showed that new neurons can restore damaged hippocampal loop.3.morris water maze test results show that the cognitive function of nt3- chitosan can improve the late injury in rats, and restore the.4.brdu/nestin characteristics of LTP Schaffer in hippocampus -ca1 collateral synapses staining showed that nt3- chitosan can activate endogenous NSCs, and promote they migrate to the injured area of.5.brdu staining showed that TBI increased the damage of ipsilateral dgbrd The number of u+ cells, nt3- chitosan increased proliferation reaction induced by TBI further, the ipsilateral nt3- chitosan group cells were more than single injury group at different time points after operation. The distribution of damage analysis results on the proliferation of DG cells showed different postoperative time points nt3- chitosan group and the single injury group brdu+ cells are still located in the neurogenesis zone SGZ, nt3- and chitosan group positive cells than single injury group, the remaining cells distributed in the DG region is non neurogenic.6.brdu/dcx molecular layer and hilus staining showed that nt3- chitosan increased the number of sgzbrdu+/dcx+ cells, while BrdU+/ increased the percentage of Dcx+ cells, indicating that NT3- chitosan not only increased the number of SGZ the new generation of nerve cells, and increase the proportion of nerve cells in the proliferation of.BrdU/NeuN/GFAP cells in immune markers showed that NT3- chitosan promotes the survival of DG cells proliferation, and increased DG in newborn granule neuronal number The amount of DG, promote the neurogenesis of.7.BrdU/Dcx staining showed that NT3- chitosan SGZ NSCs to promote the migration of.8.BrdU damage zone staining showed that NT3- chitosan increased TBI damage caused by ipsilateral pPV NSCs proliferation, BrdU/Dcx staining and Dil labeling experiments in vivo, NT3- chitosan to promote the periventricular area of NSCs migration to the damaged area the Dil+ cells in the damage zone detected expression of NSCs marker nestin, expressed as a part of the neuronal marker Dcx, indicating the periventricular area of NSCs may be involved in the damage zone of new neurons to regenerate.9.CD45 and CD11b staining results showed that NT3- chitosan inhibited the activation of macrophage infiltration in the damage zone and microglia at the same time, recovery of injury in different condition of microglia around the brain. The proportion of real-time quantitative PCR showed that the expression of NT3- TBI after chitosan inhibits proinflammatory factor IL-1 alpha beta and TNF-, at the same time The increase of anti-inflammatory factor IL-4 and IL-10 levels, the anti-inflammatory effect of chitosan NT3-.10.Reca-1 fluorescence staining showed that NT3- chitosan promoting vascular regeneration in the damage zone, damage zone increase vascular regeneration density and diameter. Conclusion: NT3- chitosan sustained release NT3 by neurotrophic factors, activate SGZ and pPV endogenous NSCs, and promote them to transfer ectopic injury area. At the same time provides a anti-inflammatory and vascular regeneration microenvironment of the bioactive materials for the damage zone, NSCs is favorable to the differentiation of migrate to the injury area as neurons mature, and the establishment of functional neural network, and then repair the damaged brain function provides a theoretical. These results and experimental basis for clinical application in the repair of NT3- chitosan bioactive material in adult brain injury.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.15;R318.08

【相似文献】