胶质细胞源性神经营养因子对脊髓损伤大鼠模型抗氧化研究

发布时间：2018-07-06 18:50

本文选题：神经因子 + 脊髓损伤　；参考：《南方医科大学》2016年博士论文

【摘要】：背景：脑和脊髓共同构成神经系统的主体部分中枢神经系统,主要负责信息的加工、传递和储存。脊髓位于脊椎骨组成的椎管内且被脊椎保护,是中枢神经的低级延伸部分,同时也是人体简单活动的初级反射中枢。交感神经和一部分副交感神经都起源于脊髓侧角或者相当于侧角的部位,主要功能是在周围神经与大脑之间传递信息。脊髓具有反射、传导、运动和调节四大功能,来自四肢、躯干和大部分内脏的各种感觉传入神经冲动,能够通过脊髓的上行纤维束传入到大脑中枢系统,进行更加高级的综合分析。由大脑发出的冲动,也必须通过脊髓白质的下行纤维束才能够调节躯干、四肢骨骼肌和内脏的生理活动。脊髓损伤(Spinal cord injury, SCI)作为一种常见的高致残性、致死性神经系统创伤,同时也是最严重的脊柱损伤并发症。外伤、炎症、肿瘤等导致椎体的移位或碎骨片突出于椎管内,导致脊髓或马尾神经出现不同程度的损伤,最终导致各种运动、感觉和括约肌功能障碍,肌张力及病理反射异常,会出现感觉障碍、运动障碍以及反射功能障碍等症状,还会导致许多器官功能障碍,其中以循环系统和呼吸系统的并发症最为普遍且常见。随着全球经济的快速发展,脊髓损伤的发生率呈现出上升趋势。脊髓损伤按照病因学、病理学、损伤的严重程度和解剖部位不同可以分成不同的类型,疾病的演变进程呈现瀑布样级联反应,目前医学界对脊髓损伤发病机制有待深入阐述。研究表明,脊髓损伤主要包括原发性损伤和继发性损伤两种损伤机制。其中,原发性损伤被动地发生在损伤后短时间内,是局限的、不可逆的。原发性损伤后的数分钟到数天内可逐渐形成可干预性的继发损害。继发性脊髓损伤的病理改变是由微循环障碍、兴奋性氨基酸毒性、自由基损伤、一氧化氮机制、细胞凋亡与坏死、钙超载、炎症反应、神经肽、内皮素、前列腺素等等共同作用的结果。有资料显示原发性损伤和继发性损伤二者均对进行性的神经组织功能丧失起到至关重要的作用。本病发病机制复杂,治疗难度大,给患者本人带来不可估量的身心伤害的同时,还给患者家庭和整个社会造成巨大的经济负担。多种直接或间接致病因素均可造成不同程度的脊髓损伤,除了物理损伤以外,氧化应激也参与中枢神经系统损伤机制的发生发展。机体在遭受任何形式的创伤时,神经因子可以触发固有干细胞作为信息传导信号。脊髓组织含有丰富的脂类物质,易感于脂质过氧化反应。生理状态下,内源性氧化系统(包括超氧化物岐化酶(Superoxide dismutase,SOD)和过氧化氢酶(Catalase, CAT)等)为了维持人体内各种细胞和亚细胞的结构完整性,可以有效地消除人体内多余的自由基。脊髓损伤后,自由基生成增加与清除障碍,脊髓组织脂质过氧化产物蓄积,细胞膜的结构、流动性和通透性遭到破坏,同时抗氧化剂水平降低和Na+-K+-ATP酶系统受抑制,脊髓组织缺血、缺氧和细胞能量代谢失常,细胞内钙超载,使细胞线粒体电子传递链脱耦联,又产生和释放大量氧自由基,最终导致神经细胞及髓鞘的结构与功能受到损害。研究认为影响脊髓损伤治疗与预后的因素主要是神经修复与再生的困难性,因此,脊髓损伤治疗的主要目的是通过各种治疗措施为脊髓损伤的神经再生提供一个有利的微环境,促进受损神经轴突的再生以恢复其功能。目前医疗界尚缺乏有效的脊髓损伤的治愈手段,针对脊髓损伤的预防、治疗和康复已成为全世界普遍面临的一个大问题。脊髓损伤患者的临床治疗只能行脊髓减压及脊椎稳定术,无法根本性修复已经损坏的脊髓组织。随着神经损伤分子病理学研究领域的不断突破,人们发现机体产生的可溶性蛋白质分子——神经营养因子(Neurotrophicfactors,NTFS),能够促进神经细胞存活、生长和分化,对于神经系统的发育及营养具有重要意义。胶质细胞源性神经营养因子(Glial derived neurotropic factor, GDNF)广泛分布于神经系统中,在神经系统的发育、生长、损伤和修复的过程中均扮演着重要的角色,对多种神经元具有营养作用,可以预防缺血性脑血管疾病和神经系统变性性疾病。GDNF已经成为当前生命科学领域的研究热点之一。目的：理论研究部分旨在通过对文献的深入研究挖掘,探讨现代医学与传统医学对脊髓损伤认识的异同及各自的特点,总结近年来脊髓损伤发病机理方面取得的成就,深入挖掘神经营养因子治疗髓损伤领域的当前研究现状,以期为今后该领域的研究提供相关数据支持。实验研究部分的主要目的是探讨胶质细胞源性神经营养因子在大鼠脊髓损伤模型中的抗氧化作用。方法：理论研究部分以PubMed为主要检索工具,按照严格的纳入排除标准,筛选出符合标准的神经营养因子治疗脊髓损伤的相关文献,通过文献计量分析方法总结脊髓损伤领域的研究现状及热点问题。实验研究部分采用Wistar大鼠静力性脊髓压迫损伤模型。健康成年雌性Wistar大鼠60只,随机分成5组：假手术组、手术对照组和给药组Group Ⅰ、Group Ⅱ、Group Ⅲ (GDNF 5mg/kg, 10mg/kg,20mg/kg),每组12只,每天经鼻饲给予GDNF 1次,连续给药5天。评估血清总抗氧化状态(Total oxidant status,TOS),检测脊髓组织中ROS水平、脂质过氧化产物和蛋白质羰基含量等指标来评价GDNF对脊髓损伤大鼠氧化应激水平的影响：测定抗氧化物谷胱甘肽(Glutathione, GSH)水平,过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(Glutathione peroxdiase,GPx)活性和谷胱甘肽-S转移(Glutathione-S-transferase,GST)反应等指标来考察GDNF的抗氧化能力。结果：文献计量学分析了从1990年至2015年25年间发表的关于神经营养因子治疗脊髓损伤的文献,共计606篇。其中以实验动物为研究对象的有408篇(占67.33%,408/606),以人为研究对象的是156篇(占25.74%,156/606)。神经营养因子治疗脊髓损伤的相关研究总体成上升趋势,该领域仍是当前研究的热点问题。截止至2015年神经营养因子治疗脊髓损伤相关研究最多的国家是美国,其次是中国,日本、加拿大、瑞典紧随其后位列第3-5位。在相关文献总发表量排名达到前10的期刊中,神经科学相关专业的期刊有9种(占90%,9/10),但只有《journal of neuroscience》的影响因子在5以上。排名前10热点关联词分别是神经生长因子(Nerve Growth Factors, NGF)、功能恢复(Recovery of Function)、神经营养因子受体结合(neurotrophin receptor binding)、神经再生(Nerve Regeneration)、干细胞(Stem Cells)、神经元可塑性(Neuronal Plasticity)、神经营养素3(Neurotrophin 3,NT-3)、BDNF、trkB受体(Receptor, trkB)、谷胱甘肽调节钾外排系统蛋白kefB (Glutathione-regulated potassium-efflux system protein kefB)。在GDNF抗脊髓损伤后氧化损伤的实验研究中发现,与模型组相比,GDNF(10mg/kg)能显著降低血清TOS,减少脊髓组织中的ROS水平,同时,GDNF可显著降低脂质过氧化和蛋白质羰基化,升高GSH、CAT、SOD、GPx、GST的水平,改善氧化应激。GDNF减少氧化应激,增加抗氧化水平,呈剂量依赖性。10mg/mL and 20mg/mL较对照组明显改善,彼此之间无显著差异。结论：本研究证实GDNF是通过减少氧化应激,增加抗氧化水平来实现对脊髓损伤大鼠模型的生物学作用。GDNF在抗脊髓损伤方面具有较好的生物学活性,值得深入探索和研究。
[Abstract]:Background: the central nervous system, the main part of the brain and spinal cord, is the main part of the nervous system, responsible for the processing, transmission and storage of information. The spinal cord is located in the spinal canal and is protected by the spine. It is the low-level extension of the central nervous system and the primary reflex center of the human body. The sympathetic nerve originates from the lateral horn of the spinal cord or the location of the lateral angle. The main function is to transmit information between the peripheral nerve and the brain. The spinal cord has four functions of reflex, conduction, motion, and regulation. The sensory afferent impulses from the limbs, trunk and most of the viscera can be afferent through the superior fiber bundle of the spinal cord to large The brain central system makes a more advanced comprehensive analysis. The impulses from the brain also have to be able to regulate the trunk, the skeletal muscles and the internal organs of the limbs through the descending fibers of the white matter of the spinal cord. Spinal cord injury (SCI) is a common high disability, fatal nervous system trauma, and also the most severe. Severe complications of spinal injury. Trauma, inflammation, and tumor, resulting in vertebral displacement or broken bone fragments out of the spinal canal, resulting in varying degrees of damage to the spinal cord or the cauda equina, resulting in various movements, sensory and sphincter dysfunction, muscular tension and pathological reflex, and the appearance of sensory disorders, dyskinesia, and reflex function. Disorders such as symptoms can also lead to many organ dysfunction, with complications most common and common in the circulatory system and the respiratory system. With the rapid development of the global economy, the incidence of spinal cord injury is on the rise. Spinal cord injury can be divided according to etiology, pathology, severity of injury and dissection of the anatomy. In the same type, the process of the evolution of the disease presents cascade cascade reaction, and the pathogenesis of spinal cord injury remains to be discussed in the medical field. The study shows that the spinal cord injury mainly includes two mechanisms of primary injury and secondary injury. The pathological changes in secondary spinal cord injury include microcirculation disorder, excitatory amino acid toxicity, free radical damage, nitric oxide, apoptosis and necrosis, calcium overload, inflammatory reaction, neuropeptides, endothelin, prostaglandins and other common effects on secondary spinal cord injury. Results. Data show that both primary and secondary injuries are of vital importance to the loss of progressive neurological function. The pathogenesis of this disease is complex, the treatment is difficult, it brings immeasurable physical and mental injury to the patient, and it also gives a huge economic burden to the patients' family and the society as a whole. Direct or indirect pathogenic factors can cause different degrees of spinal cord injury. In addition to physical damage, oxidative stress also participates in the development of the central nervous system damage mechanism. When the body suffers any form of trauma, the nerve factor can trigger the inherent stem cells as a signal transduction signal. The spinal cord contains rich lipids. Substances are susceptible to lipid peroxidation. Under physiological conditions, endogenous oxidative systems (including Superoxide dismutase, SOD) and catalase (Catalase, CAT), etc.) can effectively eliminate the excess free radicals in the human body in order to maintain the structural integrity of various cells and subcells in the human body. The lipid peroxidation product of the spinal cord, the accumulation of lipid peroxidation products in the spinal cord, the structure, the structure, the fluidity and permeability of the cell membrane were destroyed, and the level of antioxidant and the Na+-K+-ATP enzyme system were inhibited, the spinal cord ischemia, anoxia and cell energy metabolism disorder, the intracellular calcium overload, and the removal of the mitochondrial electron transfer chain. Coupled with a large number of oxygen free radicals produced and released, the structure and function of the nerve cells and myelin sheath were damaged. The study believed that the main factors affecting the treatment and prognosis of spinal cord injury were the difficulty of nerve repair and regeneration. Therefore, the main purpose of the treatment of spinal cord injury is to use various treatment measures to regenerate the nerve of spinal cord injury. It provides a favorable microenvironment to promote the regeneration of the damaged neurite to restore its function. There is still a lack of effective cure for spinal cord injury. The prevention of spinal cord injury, treatment and rehabilitation have become a major problem all over the world. The clinical treatment of spinal cord injury patients can only be treated with spinal cord decompression and spinal cord. Vertebral stabilization can not fundamentally repair the damaged spinal cord. With the continuous breakthrough in the field of molecular pathology, the soluble protein molecule Neurotrophicfactors (NTFS) produced by the body can promote the survival, growth and differentiation of nerve cells and the development of the nervous system. And nutrition is of great significance. Glial derived neurotropic factor (GDNF) is widely distributed in the nervous system and plays an important role in the development, growth, injury and repair of the nervous system. It is nutritious to a variety of neurons and can prevent ischemic cerebrovascular disease. .GDNF, a neurodegenerative disease, has become one of the hot topics in the field of life science. The purpose of this study is to explore the similarities and differences between modern medicine and traditional medicine on spinal cord injury and their respective characteristics through in-depth study of literature. In order to explore the current research status of neurotrophic factors in the field of spinal cord injury in order to provide data support for future research in this field, the main purpose of the experimental study is to explore the anti oxidative effect of glial cell derived neurotrophic factor in the rat model of spinal cord injury. Method: the theoretical research part is PubMed For the main retrieval tools, the relevant literature on the treatment of spinal cord injury with standard neurotrophic factors was selected according to the strict inclusion criteria, and the research status and hot issues in the field of spinal cord injury were summarized by bibliometric analysis. The experimental research part adopted the static spinal cord compression damage model of Wistar rats. 60 female Wistar rats were randomly divided into 5 groups: the sham operation group, the operation control group and the administration group Group I, the Group II, the Group III (GDNF 5mg/kg, 10mg/kg, 20mg/kg), 12 rats in each group, and were given GDNF 1 times by nasal feeding every day for 5 days. The serum total antioxidant status (Total oxidant status) was evaluated, and the levels of spinal cord tissue were detected and lipid levels were detected, lipids were detected, lipid levels detected in spinal cord tissue, lipid levels, lipids detected lipids levels, lipids detected in spinal tissue, lipids levels, lipids detected lipids levels, lipids detected levels, lipids in spinal tissue, lipids levels, lipids detected lipids levels, lipids detected levels, lipids levels detected spinal spinal tissue, lipid levels detected lipids levels, detected lipids in spinal tissue, lipid levels, lipids levels, lipids were detected The effects of peroxide product and protein carbonyl content on the level of oxidative stress in rats with spinal cord injury: the determination of the levels of glutathione (Glutathione, GSH), catalase (CAT), superoxide dismutase (SOD), glutathione enzyme (Glutathione peroxdiase, GPx), and glutathione -S transfer in rats with spinal cord injury (GDNF) (Glutathione-S-transferase, GST) reaction and other indicators to examine the antioxidant capacity of GDNF. Results: Bibliometrics analyzed the literature of neurotrophic factors for spinal cord injury published from 1990 to 2015, total of 606 articles. Among them, 408 (67.33%, 408/606) in experimental animals were studied for human studies. Like 156 articles (25.74%, 156/606). Neurotrophic factors in the treatment of spinal cord injury are generally on the rise, and this area is still a hot topic of current research. By the year 2015, the most research countries in the neurotrophic factor for spinal cord injury were the United States, which was followed by China, Japan, Canada and Sweden. Number 3-5. Among the first 10 journals, there were 9 journals (90%, 9/10) for neuroscience related majors, but only the factors of were more than 5. The top 10 hotspots were Nerve Growth Factors, NGF, and functional recovery (Recovery of Function). Neurotrophic factor receptor binding (neurotrophin receptor binding), nerve regeneration (Nerve Regeneration), Stem Cells, neuron plasticity (Neuronal Plasticity), neurotrophin 3 (Neurotrophin 3, NT-3), BDNF, glutathione regulated potassium excretion system Sium-efflux system protein kefB). In the experimental study of oxidative damage to GDNF after spinal cord injury, it was found that GDNF (10mg/kg) could significantly reduce serum TOS and reduce ROS levels in spinal cord tissue compared with the model group. Meanwhile, GDNF can significantly reduce lipid peroxidation and protein carbonylation and increase GSH, CAT, enrichment, and oxidation should be improved. .GDNF reduced oxidative stress and increased the level of antioxidant activity. The dose dependent.10mg/mL and 20mg/mL was significantly improved compared with the control group. There was no significant difference between each other. Conclusion: This study confirms that GDNF is a biological effect of.GDNF on spinal cord injury by reducing oxidative stress and increasing the level of antioxidant activity in the rat model of spinal cord injury. The mask has good biological activity and deserves further exploration and research.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R651.2

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