本文選題：脊髓損傷　切入點：鐵死亡　出處：《天津醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:脊髓損傷(Spinal Cord Injury,SCI)是臨床上常見的一種嚴(yán)重的疾病,具有很高的發(fā)生率并且脊髓損傷一旦發(fā)生就會導(dǎo)致非常高的致殘率,脊髓損傷后的神經(jīng)和功能恢復(fù)一直是世界性難題。因為現(xiàn)在還沒有有效的治療和康復(fù)方法,目前僅有不到1%的脊髓損傷患者達(dá)到所有神經(jīng)功能的恢復(fù)。當(dāng)前,脊髓損傷治療的瓶頸在于如何控制繼發(fā)性脊髓損傷中的大量神經(jīng)及其它細(xì)胞的死亡。因此,SCI后減小繼發(fā)性損傷的程度,減少殘存神經(jīng)細(xì)胞的死亡,這可能是脊髓損傷的治療和促進(jìn)其恢復(fù)的的關(guān)鍵所在。目前對繼發(fā)性脊髓損傷的機制仍不清楚,但是脊髓損傷后的活性氧簇(ROS)的生成和脂質(zhì)過氧化是繼發(fā)性脊髓損傷的一個非常重要的因素。并且研究還發(fā)現(xiàn)在脊髓損傷后損傷區(qū)域的細(xì)胞內(nèi)鐵過載,這大大增加了脊髓損傷后的氧化應(yīng)激壓力。最近研究表明,組織損傷后局部鐵離子濃度升高,促進(jìn)了氧自由基的產(chǎn)生,氧自由基又能夠促進(jìn)脂質(zhì)過氧化,導(dǎo)致了周圍組織和細(xì)胞的損傷。先前研究表明,鐵螯合劑和一些抗氧化藥物在脊髓損傷修復(fù)中具有一定的保護作用。然而,其機制并不清楚,制約了在臨床治療脊髓損傷的應(yīng)用。根據(jù)現(xiàn)有繼發(fā)脊髓損傷階段的研究,近年發(fā)現(xiàn)的一種新的程序性死亡機制-鐵死亡(Ferroptosis)可能在脊髓損傷中存在,并且鐵死亡抑制劑可能能夠保護脊髓損傷并且促進(jìn)其修復(fù)。鐵死亡在缺血再灌注型組織損傷中普遍存在,鐵死亡抑制劑可以顯著修復(fù)組織損傷。由于脊髓損傷中鐵的超載,以及脂質(zhì)過氧化,提示可能存在鐵死亡途徑。本課題聚焦鐵死亡在脊髓損傷中的作用和機制,試圖通過鐵死亡抑制劑干預(yù)鐵死亡,從而修復(fù)脊髓損傷。本研究的核心問題是:鐵死亡在繼發(fā)性脊髓損傷中的作用,阻斷這一死亡途徑是否可以實現(xiàn)脊髓損傷的修復(fù)。因此,本課題設(shè)計如下:(1)驗證脊髓損傷中是否存在鐵死亡;(2)鐵死亡特異性抑制劑是否對脊髓損傷和神經(jīng)元細(xì)胞損傷具有保護作用及其機制。方法:體內(nèi)實驗證明鐵死亡存在于大鼠脊髓損傷中且可被鐵死亡抑制劑干預(yù)。建立Wistar大鼠脊髓損傷挫傷模型,并對損傷大鼠一次性給與鐵死亡抑制劑SRS16-86,BBB評分評價鐵死亡抑制劑對大鼠脊髓損傷后運動功能的保護作用,免疫組織化學(xué)染色觀察神經(jīng)元細(xì)胞存活數(shù)量、膠質(zhì)瘢痕和脊髓空洞面積,從而評價鐵死亡抑制劑對脊髓損傷的保護作用。檢測鐵死亡標(biāo)志物,證明脊髓損傷中存在鐵死亡。本實驗應(yīng)用大鼠脊髓挫傷模型,隨機分為SRS 16-86處理組、對照組、假手術(shù)組,并且在不同的時間點進(jìn)行大鼠后肢功能評定(BBB評分),評價鐵死亡抑制劑(SRS 16-86)對大鼠脊髓損傷的保護作用。檢測ROS代謝產(chǎn)物4HNE的含量以及GPX4的表達(dá)。檢測脊髓中鐵離子含量,評價鐵離子和脊髓損傷嚴(yán)重程度相關(guān)性。免疫熒光染色觀察損傷脊髓中星型膠質(zhì)細(xì)胞、神經(jīng)元和少突膠質(zhì)細(xì)胞。結(jié)果:成功建立大鼠脊髓損傷挫傷模型,并通過組織病理學(xué)和功能學(xué)評分進(jìn)行驗證;成功自行合成了鐵死亡的抑制劑SRS 16-86,并通過質(zhì)譜分析驗證有效;使用SRS 16-86干預(yù)脊髓損傷能夠減少ROS代謝產(chǎn)物4HNE的含量,增加損傷區(qū)域的總谷胱甘肽,減少了損傷區(qū)域的鐵離子含量,并且使用SRS 16-86干預(yù)后改善脊髓損傷大鼠的行為學(xué)評分,以及改善組織病理學(xué)變化,抑制星形膠質(zhì)細(xì)胞的增生,增加神經(jīng)元標(biāo)志物的表達(dá),增加CNPase陽性區(qū)域的面積;并且SRS16-86還能夠減少脊髓損傷區(qū)域TNF-α、IL-1β和ICAM-1的表達(dá),抑制脊髓損傷后的炎癥反應(yīng)。結(jié)論:本研究初步證實在脊髓損傷中存在鐵死亡這種程序性細(xì)胞死亡方式;SRS16-86能夠抑制鐵死亡,并且顯著改善脊髓損傷后的后肢運動功能,改善組織病理學(xué)變化,保護了殘存的神經(jīng)細(xì)胞和保護少突膠質(zhì)細(xì)胞,減少了后期星形膠質(zhì)細(xì)胞的增生以及膠質(zhì)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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