【作者】 于继徐；

【导师】 李春岩；

【作者基本信息】 河北医科大学 ， 神经病学， 2009， 博士

【摘要】 肌萎缩侧索硬化(ALS)是中枢神经系统常见的一种慢性神经退行性疾病,以脑和脊髓中选择性的运动神经元变性为特征,临床表现为缓慢起病,进行性发展。ALS是一种致死性疾病,患者多在首次出现症状后的3~5年内死亡。目前,该病病因不清、发病机制不明、缺乏有效的治疗方法。目前证据提示ALS运动神经元的丢失是由于一些复杂的相互作用的机制所致,包括氧化应激、兴奋性毒性作用、细胞骨架异常和蛋白聚集以及遗传因素等。诸多研究表明这些机制之间并不是相互排斥的,在最终导致ALS的运动神经元死亡的不同机制中氧化应激可能起中心作用。氧化应激是指活性氧簇(ROS)和活性氮簇(RNS)及其代谢产物产生过多引起的细胞损伤。神经系统氧耗量大,多不饱和脂肪酸含量丰富,抗氧化能力相对较弱。与其他器官相比,神经系统更易受到氧化损伤。中枢神经系统含有非血红素铁,并且在特定部位如黑质和基底节中铁含量非常丰富。诸多研究表明,铁与氧化应激关系密切。铁离子如果不能以适当方式与蛋白或其它配体结合,其可以通过Fenton反应催化形成具有代谢毒性的ROS,攻击生物大分子,从而引起神经元变性。大量临床研究发现,在多种神经退行性疾病中都存在氧化应激和异常铁积聚的现象,如包括帕金森病和阿尔茨海默病在内多种神经退行性疾病病人脑内呈现病理性、区域性铁沉积。据此我们推测,铁过载导致的氧化应激在ALS的发病中可能发挥重要作用。我们应用谷氨酸转运体抑制剂苏-羟天冬氨酸(THA)诱导的脊髓体外器官型培养谷氨酸兴奋性毒性模型,探讨铁在谷氨酸兴奋性毒性中的作用,以及绿茶多酚EGCG和莱菔硫烷(SF)对运动神经元的作用和机制。采用免疫组织化学方法观察运动神经元的数目和形态,用Western blot方法检测标本蛋白水平的变化,用多功能酶标仪检测乳酸脱氢酶、丙二醛和谷氨酸的变化。研究发现,在谷氨酸兴奋性毒性模型中,转铁蛋白受体和二价金属离子转运体蛋白表达升高,铁蛋白表达降低,组织内总铁含量增加,氧化应激水平升高,运动神经元数目减少,给予铁螯合剂去铁敏可以有效阻止THA导致的氧化应激,保护运动神经元。绿茶多酚EGCG作用机制复杂,我们研究表明,EGCG可以通过降低细胞间隙的谷氨酸来对抗THA引起的兴奋性毒性,减轻氧化应激,保护运动神经元。莱菔硫烷是一个非常有效的Ⅱ相酶诱导剂,在肿瘤研究中,诸多学者对其进行了深入地探讨。我们发现莱菔硫烷可以通过诱导Ⅱ相解毒酶有效对抗THA引起的运动神经元死亡。另外,我们对谷氨酸兴奋性毒性模型进行了深入地分析研究,为进一步应用此模型进行实验研究夯实了基础。通过上述一系列实验研究,我们阐明了铁在谷氨酸兴奋性毒性中的重要作用以及EGCG和莱菔硫烷通过不同的机制保护运动神经元。这对于研究肌萎缩侧索硬化的发病机制和筛选有效的治疗药物提供了坚实的理论依据。第一部分铁在谷氨酸兴奋性毒性中的重要作用目的:在脊髓体外器官型培养谷氨酸兴奋性毒性模型中,观察铁转运、储存蛋白和铁含量的变化,并且应用铁螯合剂去铁敏,观察其对运动神经元是否具有保护作用。方法:应用脊髓体外器官型培养模型,随即分成3组:对照组、THA组和DFO+THA组。应用Western blot方法检测铁转运和储存蛋白水平的变化,用原子吸收光谱法测量组织中铁含量的变化,用免疫组化方法检测运动神经元数目,用多功能酶标仪检测组织中丙二醛的变化以及培养液中谷氨酸和乳酸脱氢酶的变化。结果:100μmol/L THA处理3周后,TfR、DMT1-IRE和DMT1+IRE蛋白表达升高,而Ft-H蛋白表达下降,组织中总铁含量增加。给予铁螯合剂DFO可以有效阻止THA引起的兴奋性毒性,保护运动神经元。但是DFO对于培养液中的谷氨酸没有影响。结论:铁在谷氨酸兴奋性毒性中起到重要作用,螯合铁可以有效阻止谷氨酸兴奋性毒性引起的运动神经元损伤。第二部分绿茶多酚EGCG对运动神经元的保护机制探讨目的:探讨绿茶多酚EGCG对运动神经元的作用和机制,为寻找肌萎缩侧索硬化的治疗药物提供理论依据。方法:应用脊髓体外器官型培养模型,随机分成3组:对照组、THA组和EGCG+THA组。用免疫组化方法检测运动神经元数目,用Westernblot方法检测Ⅱ相酶蛋白水平的变化,用多功能酶标仪检测组织中丙二醛的变化以及培养液中谷氨酸和乳酸脱氢酶的变化。结果:100μmol/L THA干预后运动神经元数目较正常对照组明显减少(P<0.05),而用5μmol/L EGCG预处理48小时,再给予5μmol/L EGCG+100μmol/L THA联合处理3周后运动神经元数目较THA组明显增加(P<0.05)。EGCG+THA组培养液中谷氨酸的含量较THA组明显降低,同时检测MDA水平也显著下降(P<0.05)。与Ⅱ相酶诱导剂SF保护运动神经元不同,EGCG+THA处理3周后Ⅱ相解毒酶NQO-1和HO-1表达较THA组下降。结论:EGCG通过降低细胞间隙的谷氨酸,可以阻止THA引起的兴奋性神经毒性,保护运动神经元。第三部分莱菔硫烷诱导Ⅱ相酶表达保护大鼠脊髓运动神经元目的:探讨Ⅱ相酶诱导剂莱菔硫烷(SF)对运动神经元的作用和机制。方法:应用SD乳鼠脊髓体外器官型培养模型,随机分成3组:对照组、THA组和SF+THA组。用免疫组化法检测运动神经元数目,用Western blot法检测Ⅱ相酶蛋白水平,用多功能酶标仪检测培养液中谷氨酸的变化。结果:THA干预后运动神经元数目较对照组明显减少(P<0.05),而应用SF预处理48 h,再给予SF和THA联合处理3周后运动神经元数目较THA组明显增加(P<0.05),同时Ⅱ相酶NQO-1和HO-1表达明显升高(P<0.05)。结论:SF通过诱导Ⅱ相酶NQO-1和HO-1的表达,可以有效预防THA引起的运动神经元损伤。第四部分谷氨酸兴奋性毒性模型的研究目的:探讨谷氨酸兴奋性毒性模型-THA诱导的脊髓器官型培养模型培养液各成分中谷氨酸的含量及模型培养前后培养液中谷氨酸的变化。方法:应用脊髓体外器官型培养模型,随机分成2组:对照组、THA组。用免疫组化法检测运动神经元数目,用多功能酶标仪检测培养基中谷氨酸的含量。结果:最小基础培养基(MEM)和马血清(HS)中均含有谷氨酸。经过4周培养后,THA组培养液中谷氨酸的含量明显高于对照组,运动神经元数目较对照组减少。结论:THA诱导的脊髓器官型培养模型作为谷氨酸兴奋性毒性模型,我们在应用它时一定要严格检测培养液中谷氨酸的含量,对于每一个处理因素都要考虑其对谷氨酸的影响。更多还原

【Abstract】 Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and lethal neurodegenerative disease, characterizing by the degeneration of motor neurons from cortex, brainstem, and spinal cord. ALS is one of the most common neurodegenerative disorders, occurring both sporadically (sALS) and as a familial disorder (fALS) with inherited cases accounting for about 10% of patients. Using the current standard therapy, the typical survival time for patients after diagnosis is three years, although large deviation has been observed. Present evidence indicates that loss of neurons in ALS results from a complex interplay among oxidative injury, excitotoxic stimulation, aggregation and/or dysfunction of critical proteins and genetic factors. Recent investigations support that these mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress.Reactive oxygen species (ROS), such as superoxide anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH?), are continuously generated during oxidative metabolism in biological systems. ROS are prominent toxic intermediates, potentially damaging all types of biological molecules. Although cellular antioxidants act in concert to detoxify these species, an increase in the production of ROS and/or a decrease in the antioxidant capacity of cells can cause oxidative stress.CNS is particularly vulnerable to oxidative damage, due to a high level of oxidative capacity, high concentrations of polyunsaturated fatty acids. In addition, the CNS contains non-haem iron and certain brain structures are particularly rich in iron.Iron is essential for normal physiology, and it is implicated in many pathological processes, including neuron degenerative disorders. Iron is known to play an important role in Alzheimer disease, including accelerating amyloid-βaggregation and promoting oxidative damage. We suspected that iron might play an important role in glutamate excitotoxicity and neuronal death. Iron is known to potentiate the toxic effects of ROS by catalyzing the formation of highly reactive hydroxyl radicals from hydrogen peroxide through the Fenton chemistry.In the present study, we studied effect of iron on glutamate excitotoxicity and the strategies to protect motor neurons. We measured tissue iron levels and tissue levels of transferrin receptor, divalent metal-ion transporter 1 and ferritin in organotypic culture of rat spinal cord with and without THA-induced glutamate excitotoxicity. Moreover, the role of iron in glutamate excitotoxicity was assessed by measuring the protective activity of iron chelator deferoxamine. Moreover, we studied the mechanisms of EGCG and sulforaphane protects motor neurons in organotypic culture of rat spinal cord.We found: 1. THA could increase tissue iron level in the spinal cord tissue, with concomitant modulation of several iron transport and storage proteins, including transferrin receptor, divalent metal-ion transporter 1 and ferritin. More significantly, iron chelator deferoxamine was able to prevent THA-induced motor neuron degeneration completely. 2. EGCG could protect motor neurons via regulating glutamate levels in organotypic culture of rat spinal cord. 3. SF could prevent motor neuron death caused by THA toxicity via inducing the expression of phaseⅡenzymes.Part I Iron is a potential key mediator of glutamate excitotoxicity in spinal cord motor neuronsObjective:To study effect of iron on glutamate excitotoxicity. Methods:Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 7-day-old rat pups. The cultures were divided into three groups at random: control, 100μmol/L THA group, DFO plus THA group. The number of motor neurons was assessed by immunohistochemistry, the expression of transferrin receptor, divalent metal-ion transporter 1 and ferritin were assayed with western blot, the level of lactate dehydrogenase, malondialdehyde and glutamate was assayed with ELIASA, the iron level in the explants was assayed with atomic absorption spectrometry.Results:At the end of the 3-week THA treatment, we found that the expression levels of TfR, DMT-IRE and DMT1+IRE were all increased significantly after THA treatment, whereas the level of ferritin decreased significantly, total iron content in the explants increased 21.4%. DFO prevented THA-induced neuron death, the number of motor neurons per ventral horn was higher in explants treated with 100μmol/L DFO plus 100μmol/L THA than in the control. Moreover, DFO could prevent the increase of lactate dehydrogenase and malondialdehyde induced by THA. But DFO had no effect on the medium glutamate.Conclusion: Iron is a potential key mediator of motor neuron excitotoxicity in organotypic culture of rat spinal cord. Blocking THA-induced iron rise alone may be sufficient for prevention of glutamate excitotoxicity.PartⅡThe mechanisms of green tea polyphenol protecting motor neuronsObjective: To study the mechanisms of green tea polyphenol, EGCG, protecting motor neurons in organotypic culture of rat spinal cord. Methods: The SD rat pups spinal cord organotypic cultures were divided into three groups at random: control, 100μmol/L THA group, EGCG plus THA group. The number of motor neurons was assessed by immunohistochemistry, and the expression of phaseⅡenzymes and EAAT2 were assayed with western blot, the level of malondialdehyde and glutamate was assayed with ELIASA.Results: At the end of 3-week treatment, the motor neurons number in the group treated with THA was less than the control group (P<0.05). However, the motor neurons number in the group treated with 5μmol/L EGCG plus 100μmol/L THA was more than group treated with THA at the end of 3 weeks (P<0.05). 5μmol/L EGCG could prevent the increase of glutamate induced by THA (P<0.05). The level of MDA decreased after treated with 5μmol/L EGCG plus 100μmol/L THA. Interestingly, the phaseⅡenzymes (NQO-1 and HO-1) in the group treated with 5μmol/L EGCG plus 100μmol/L THA expressed less than group treated with THA only.Conclusions: EGCG could prevent motor neuron death caused by THA toxicity via decreasing the synaptic cleft glutamate level in organotypic culture of rat spinal cord.PartⅢSulforaphane protects motor neurons of rat spinal cord via inducing phase II enzymesObjective: To investigate whether sulforaphane (SF) protects motor neurons via inducing phaseⅡenzymes.Methods: The SD rat pups spinal cord organotypic cultures were divided into three groups at random: control, 100μmol/L THA group, SF plus THA group. The number of motor neurons was assessed by immunohistochemistry, the expression of phaseⅡenzymes were assayed with western blot, and he level of glutamate was assayed with ELIASA.Results: At the end of 3-week treatment, the motor neurons number in the group treated with THA was less than the control group (P<0.05). However, the motor neurons number in the group treated with 10μmol/L SF plus 100μmol/L THA was more than group treated with THA at the end of 3 weeks (P<0.05). Meanwhile, the phaseⅡenzymes (NQO-1 and HO-1) in the group treated with 10μmol/L SF plus 100μmol/L THA expressed more than group treated with THA only (P<0.05). But SF had no effect on the medium glutamate.Conclusions: SF could prevent motor neuron death caused by THA toxicity via inducing the expression of phaseⅡenzymes.PartⅣStudy the model of glutamate excitotoxicityObjective: To study the levels of glutamate in medium of THA-induced spinal cord organotypic culture.Methods: The SD rat pups spinal cord organotypic cultures were divided into two groups at random: control and 100μmol/L THA group. The number of motor neurons was assessed by immunohistochemistry and the level of glutamate was assayed with ELIASA.Results: There is glutamate in minimal essential medium and horse serum. At the end of the 3-week THA treatment, we found that the medium glutamate level treated with THA is higher than the control, and the motor neurons number in the group treated with THA was less than the control group.Conclusions: It is important to assay the medium glutamate level in THA-induced spinal cord organotypic culture, and perfect results acquired from this model should be include the effect of treatment on the medium glutamate.更多还原