铁死亡在脊髓损伤中的作用及SRS 16-86的干预

发布时间：2018-03-14 04:48

本文选题：脊髓损伤　切入点：铁死亡　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:脊髓损伤(Spinal Cord Injury,SCI)是临床上常见的一种严重的疾病,具有很高的发生率并且脊髓损伤一旦发生就会导致非常高的致残率,脊髓损伤后的神经和功能恢复一直是世界性难题。因为现在还没有有效的治疗和康复方法,目前仅有不到1%的脊髓损伤患者达到所有神经功能的恢复。当前,脊髓损伤治疗的瓶颈在于如何控制继发性脊髓损伤中的大量神经及其它细胞的死亡。因此,SCI后减小继发性损伤的程度,减少残存神经细胞的死亡,这可能是脊髓损伤的治疗和促进其恢复的的关键所在。目前对继发性脊髓损伤的机制仍不清楚,但是脊髓损伤后的活性氧簇(ROS)的生成和脂质过氧化是继发性脊髓损伤的一个非常重要的因素。并且研究还发现在脊髓损伤后损伤区域的细胞内铁过载,这大大增加了脊髓损伤后的氧化应激压力。最近研究表明,组织损伤后局部铁离子浓度升高,促进了氧自由基的产生,氧自由基又能够促进脂质过氧化,导致了周围组织和细胞的损伤。先前研究表明,铁螯合剂和一些抗氧化药物在脊髓损伤修复中具有一定的保护作用。然而,其机制并不清楚,制约了在临床治疗脊髓损伤的应用。根据现有继发脊髓损伤阶段的研究,近年发现的一种新的程序性死亡机制-铁死亡(Ferroptosis)可能在脊髓损伤中存在,并且铁死亡抑制剂可能能够保护脊髓损伤并且促进其修复。铁死亡在缺血再灌注型组织损伤中普遍存在,铁死亡抑制剂可以显著修复组织损伤。由于脊髓损伤中铁的超载,以及脂质过氧化,提示可能存在铁死亡途径。本课题聚焦铁死亡在脊髓损伤中的作用和机制,试图通过铁死亡抑制剂干预铁死亡,从而修复脊髓损伤。本研究的核心问题是:铁死亡在继发性脊髓损伤中的作用,阻断这一死亡途径是否可以实现脊髓损伤的修复。因此,本课题设计如下:(1)验证脊髓损伤中是否存在铁死亡;(2)铁死亡特异性抑制剂是否对脊髓损伤和神经元细胞损伤具有保护作用及其机制。方法:体内实验证明铁死亡存在于大鼠脊髓损伤中且可被铁死亡抑制剂干预。建立Wistar大鼠脊髓损伤挫伤模型,并对损伤大鼠一次性给与铁死亡抑制剂SRS16-86,BBB评分评价铁死亡抑制剂对大鼠脊髓损伤后运动功能的保护作用,免疫组织化学染色观察神经元细胞存活数量、胶质瘢痕和脊髓空洞面积,从而评价铁死亡抑制剂对脊髓损伤的保护作用。检测铁死亡标志物,证明脊髓损伤中存在铁死亡。本实验应用大鼠脊髓挫伤模型,随机分为SRS 16-86处理组、对照组、假手术组,并且在不同的时间点进行大鼠后肢功能评定(BBB评分),评价铁死亡抑制剂(SRS 16-86)对大鼠脊髓损伤的保护作用。检测ROS代谢产物4HNE的含量以及GPX4的表达。检测脊髓中铁离子含量,评价铁离子和脊髓损伤严重程度相关性。免疫荧光染色观察损伤脊髓中星型胶质细胞、神经元和少突胶质细胞。结果:成功建立大鼠脊髓损伤挫伤模型,并通过组织病理学和功能学评分进行验证;成功自行合成了铁死亡的抑制剂SRS 16-86,并通过质谱分析验证有效;使用SRS 16-86干预脊髓损伤能够减少ROS代谢产物4HNE的含量,增加损伤区域的总谷胱甘肽,减少了损伤区域的铁离子含量,并且使用SRS 16-86干预后改善脊髓损伤大鼠的行为学评分,以及改善组织病理学变化,抑制星形胶质细胞的增生,增加神经元标志物的表达,增加CNPase阳性区域的面积;并且SRS16-86还能够减少脊髓损伤区域TNF-α、IL-1β和ICAM-1的表达,抑制脊髓损伤后的炎症反应。结论:本研究初步证实在脊髓损伤中存在铁死亡这种程序性细胞死亡方式;SRS16-86能够抑制铁死亡,并且显著改善脊髓损伤后的后肢运动功能,改善组织病理学变化,保护了残存的神经细胞和保护少突胶质细胞,减少了后期星形胶质细胞的增生以及胶质瘢痕的形成。
[Abstract]:Objective: spinal cord injury (Spinal Cord, Injury, SCI) is a serious disease, with the incidence of spinal cord injury and the event will lead to very high disability rate is very high, after spinal cord injury and nerve function recovery has been a worldwide problem. Because there is no treatment and rehabilitation method effective, currently only less than 1% of all patients with spinal cord injury to nerve function recovery. At present, the bottleneck in the treatment of spinal cord injury is how to control a large number of nerve secondary injury of spinal cord and other cell death. Therefore, SCI reduced the degree of secondary injury, reduce residual nerve cell death, which may be the treatment of spinal cord injury and promote the recovery of the key. The mechanism of secondary spinal cord injury is still unclear, but ROS after spinal cord injury (ROS) generation and lipid peroxidation Is a very important factor in secondary spinal cord injury. And the study also found that the intracellular iron overload in the region after spinal cord injury, which greatly increases the oxidative stress after spinal cord injury. Recent studies have shown that tissue damage after local iron ion concentration, promote the production of oxygen free radicals, oxygen free radicals can promote lipid peroxidation, leading to the surrounding tissue and cell injury. Previous studies showed that iron chelator and some antioxidant drugs in the repair of spinal cord injury has a protective effect. However, its mechanism is not clear, which restricts the application in the clinical treatment of spinal cord injury. According to the existing research stage secondary spinal cord injury in recent years, the discovery of a new procedural mechanism of death - iron death (Ferroptosis) may exist in spinal cord injury, and iron death inhibitors may protect spinal cord injury and To promote its repair. Iron death in ischemia reperfusion injury of common type, iron death inhibitors can significantly repair tissue damage due to overload. In spinal cord injury, and lipid peroxidation, suggesting iron death pathway. The effect and mechanism of the focal iron death in spinal cord injuries in the attempt by iron the death of iron inhibitors interfere with death, to repair spinal cord injury. The key issue of this research is: Iron death in spinal cord injury in the role of blocking whether the death pathway can be achieved in the repair of spinal cord injury. Therefore, the design of this subject are as follow: (1) verify the existence of iron death in spinal cord injury (2); iron death specific inhibitors on cell injury of spinal cord injury and has the neuroprotective effect and its mechanism. Methods: in vivo experiments showed that the iron death exists in rats with spinal cord injury and death inhibition by iron Preparation of intervention. The establishment of Wistar rat model of contusive spinal cord injury, and injury of rats given one-time iron death inhibitor SRS16-86, BBB score to evaluate the protective effect of the death of iron inhibitors on motor function in rats after spinal cord injury, immunohistochemical staining and cell survival number of neurons, glial scar and syringomyelia area, to evaluate the death of iron inhibitors on spinal cord injury. The detection of iron death markers, prove the existence of death iron cord injury. The spinal cord contusion in rats models were randomly divided into SRS treatment group 16-86, control group, sham operation group, and evaluation of rat hindlimb function at different time points (BBB score), evaluation iron death inhibitor (SRS 16-86) protective effect on spinal cord injury in rats. Detect the expression of ROS metabolites content of 4HNE and GPX4. The spinal cord containing iron ion detection, evaluation of iron The severity of spinal cord injury and spinal cord injury were observed. Immunofluorescence staining in glial cells, neurons and oligodendrocytes. Results: we successfully established the rat model of spinal cord injury and contusion, were verified by histopathology and function; successfully synthesized iron death inhibitor SRS 16-86, and analyzed by mass spectrometry verify the use of SRS 16-86; intervention of spinal cord injury can reduce the content of ROS metabolites of 4HNE, increase the total glutathione in the damaged region, reduce the amount of iron in the damaged region, and use the SRS 16-86 stem and improve the prognosis of spinal cord injury in rats and improve the behavioral score, histopathological changes, inhibit the proliferation of astrocytes. Increased neuronal marker expression, increase CNPase positive area of SRS16-86; and also can reduce the spinal cord injury area of TNF- alpha, IL-1 beta and ICAM-1 The expression of inflammatory reaction after spinal cord injury. Conclusion: This study confirmed the existence of the death of iron programmed cell death in spinal cord injury; SRS16-86 can inhibit the death of iron, and significantly improve the hind limb motor function after spinal cord injury, improve pathological changes, protect nerve cells and protect the survival of oligodendrocytes glial cells, reduced the proliferation of astrocytes and the formation of glial scar.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.2

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