【作者】 贾亚琼；

【导师】 田新英； 李春岩；

【作者基本信息】 河北医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的:肌萎缩侧索硬化(amyotrophic lateral sclerosis, ALS)是一种少见但致死性的神经系统变性疾病,它选择性侵犯大脑皮层、脊髓和脑干的运动神经元。临床表现为缓慢进展的四肢无力,逐渐丧失运动功能,最终累及呼吸肌,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、缺乏切实有效的治疗方法。大量证据提示ALS运动神经元的选择性丢失并不是单一因素造成,而是由于多种因素复杂的相互作用所致,包括氧化应激,谷氨酸兴奋毒性,遗传因素,自身免疫异常,细胞骨架异常以及蛋白质异常聚积等。而氧化应激则是其各种不同机制的最后共同通路。目前ALS的治疗主要包括兴奋性氨基酸拮抗剂力鲁唑(riluzole)、抗氧化剂、神经营养因子、基因治疗、免疫治疗以及干细胞移植治疗等方面。其中,兴奋性氨基酸拮抗剂力鲁唑是唯一用于临床有效的药物,也是唯一被美国FDA批准用于ALS临床治疗的药物,但它也只能轻微延缓疾病的进展(延长寿命90天),而对运动功能、肌力和运动症状的改善无任何作用,并且价格昂贵,非一般家庭所能承受。因此,对于ALS患者来说,亟需一种价格低廉又切实有效的药物。绿茶是一种流行世界的饮料,历来被认为是一种有益健康的饮品。近来研究表明,绿茶发挥有益作用的是绿茶叶中的一类多酚类物质,它们都属于黄酮类,合称绿茶多酚,主要包括四种有效成份:表没食子儿茶素没食子酸酯( epigallocatechin gallate,EGCG)、表儿茶素没食子酸酯(epicatechin gallate,ECG)、表没食子儿茶素(epigallocatechin,EGC)和表茶素(epicatechin,EC)。其中EGCG含量最高,大约占到绿茶提取物干重的10%,具有抗凋亡、抗癌、抗突变、抗炎等作用。近年来被广泛研究,证实其还可在多个靶点发挥神经保护作用,如自由基清除、金属螯合、影响细胞存活和凋亡基因的表达、以及对细胞信号转导通路、线粒体功能和泛素-蛋白酶体系统的调节等。之前,我们应用谷氨酸转运体抑制剂苏-羟天冬氨酸(threohydroxyaspartate,THA)诱导细胞间隙内谷氨酸浓度升高,造成谷氨酸兴奋性毒性脊髓体外器官型培养模型,用EGCG预处理,发现EGCG不仅能够保护运动神经元,减轻细胞损伤程度,减低细胞脂质过氧化水平,还能降低细胞间隙的谷氨酸浓度。这是一个非常令人振奋的结果,因为EGCG兼具了抗氧化和调节谷氨酸的双重作用。但是也正因为EGCG还具有抗氧化作用,严格来说,我们并不能确定其调节谷氨酸的作用是否是抗氧化作用的一个附加结果。N-乙酰半胱氨酸(N-acetyl cysteine,NAC)是一种广为人知的抗氧化剂,它不仅具有直接的抗氧化作用,还能为谷胱甘肽(glutathione,GSH)的合成提供底物,而谷胱甘肽则是活性氧族(reactive oxygen species,ROS)和脂质过氧化产物的清除剂。因此,为了进一步确证EGCG的这个结果,本研究引入了NAC作为参照,观察其在完全相同的体外培养模型中对运动神经元的保护作用及其对细胞间隙谷氨酸浓度的影响。方法:实验动物选用7日龄的SD(Sprague-Dawley)乳鼠快速断头,在无菌条件下迅速取出脊髓,用活组织切片机切成350μm厚的薄片,种植在6孔培养板内的插入式培养皿(30mm Millicell-CM, Millipore公司)中,每孔5片,放入二氧化碳培养箱(37.0℃,5% CO2+95%空气),每周更换2次培养液。经过1周的损伤恢复期后,在培养液中加入谷氨酸转运体抑制剂苏-羟天冬氨酸(threohydroxyaspartate,THA),使细胞间隙谷氨酸浓度增加,造成谷氨酸兴奋性毒性脊髓体外器官型培养模型。之后,脊髓片随机分为正常对照组、THA模型组和NAC提前干预组,继续培养3周后收集标本及培养液。用神经元的特异性抗体SMI-32组化染色,对脊髓前角运动神经元进行鉴定、计数,并测定培养液中的乳酸脱氢酶(lactate dehydrogenase,LDH)活力以判断细胞损伤程度,测定培养液中的谷氨酸(glutamate)浓度以判断NAC能否调节谷氨酸,测定脊髓组织中的丙二醛(malondialdehyde,MDA)含量以判断细胞脂质过氧化水平。结果:正常对照组脊髓片体外生长良好,面积逐渐增大,两侧前角共有24.13±3.44个运动神经元。THA模型组脊髓片体外生长不良,与对照组相比,前角运动神经元数明显减少,仅6.27±1.62个(P<0.05),且培养液中谷氨酸浓度,LDH活力明显增加(P<0.05),脊髓组织中MDA含量明显增加(P<0.05)。而NAC提前干预组脊髓片生长与正常对照组相近,与模型组相比前角运动神经元数目明显增加,19.29±2.13个(P<0.05),且培养液中LDH活力明显下降(P<0.05),脊髓组织中MDA含量明显减少(P<0.05),但培养液中的谷氨酸浓度无明显变化(P>0.05)。结论:抗氧化剂NAC在谷氨酸兴奋性毒性脊髓体外器官型培养模型中能够保护运动神经元,减轻细胞损伤程度,减低细胞脂质过氧化水平,但并不能直接调节细胞间隙的谷氨酸水平,因此,推测EGCG对于细胞间隙谷氨酸浓度的调节作用并不是其抗氧化作用的附加结果,而是EGCG本身即具有调节谷氨酸的作用。更多还原

【Abstract】 Objective:Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and lethal neurodegenerative disease, characterized by the degeneration of motor neurons from cerebral 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 3 to 5 years, although large deviation has been observed. Present evidence indicates that loss of neurons in ALS could not be due to a single factor,but results from a complex interplay among oxidative stress, glutamate excitotoxicity, dysfunction of autoimmunity, genetic factors,aggregation and/or dysfunction of critical proteins. Recent investigations support that among these mechanisms,oxidative stress is a common final pathway.So far,an effective disease-modifying treatment for this disabling and fatal disease is lacking.That is,proven therapeutic options are limited to riluzole,a glutamate release inhibiting drug that only extends the survival in humans by approximately 3 months without showing any therapeutic effect on motor function,muscle strength or motor symptoms.Hence,the development of innovative and more effective neuroprotective strategies for this devastating disease is of utmost importance.Green tea is a world wide beverage.It has been used in the oriental countries for a long time and traditionally thought to have diverse effects.Recently,it has been demonstrated that green tea contains many compounds that have many pharmacologic activities..Flavonoids,major polyphenolic compounds found in green tea, including epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC) and epicatechin gallate (ECG) ,protect neuronal cells from oxidative stress.EGCG is one of the most well-known green tea polyphenolics (GTPs) with diverse effects,such as anti-apoptotic, anti-cancer, anti- mutagenic,and anti-inflamentory effects.The exact mechanisms of neuroprotective effect of EGCG have not been clearly understood.Previous studies suggest that EGCG protect neuronal cells via free radical scavenging,metal chelation,the enhancing expression of cell survival gene and inhibition of apoptotic gene,the regulation of cell signal transduction pathway,mitochondrial function,and ubiquitin-protesome system.Previously,in the threohydroxyaspartate (THA) induced glutamate exitotoxicity organotypic spinal cord culture model,we pretreated explants with EGCG, and then discovered that EGCG could not only protect motor neurons,alleviate the extent of cell injury and the level of lipoperoxidation,but also regulate the glutamate concentration in the culture medium.This is an exiciting result,because in the in vivo exitotoxicity model,EGCG have both anti-oxidative and glutamate regulating effects.However,owing to the simultaneous antioxidative effect of EGCG,we do not know whether the regulation of glutamate of EGCG is related to its anti-oxidative effect.N-acetyl cysteine (NAC) is a well-known antioxidative agent.It plays antioxidative role directly or provides substrate to synthesis of glutathione (GSH),which is a potent scanvenging agent of reactive oxygen species (ROS) and lipoperoxidative products.So,in the current study,the protection to motor neurons of NAC and the mechanisms involved in are tested.Methods:Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 7-day-old rat pups.Lumber spinal cords were collected under sterile condition and sectioned transversly at 350μm intervals with a McIIwain tissure chopper.Slices were carefully placed on the surface of Millipore Millicell-CM insert (five slices per membrane).Cultures were incubated at 37℃in the 5%CO2 and 95% humidified environment.To establish the glutamate exitotoxicity spinal cord organotypic culture model,the culture medium were continuously added with 100μmol/L THA,an inhibitor of glutamate transporter.After one week of recovery,explants were treated with 100μmol/L NAC for 48 hours,and then the combination of 100μmol/L THA and 100μmol/L NAC for 3 weeks.Then collect the explants and culture medium to be tested.Motor neurons in the ventral horn were identified and evaluated by SMI-32 immunohistochemical staining.The lactate- dehydrogenase (LDH) activity in the culture medium was assayed to evaluate the extent of cell injury.The malondialdehyde (MDA) content in the spinal cord explants were assayed to evaluate the level of lipoperoxidation.The glutamate concentration in the culture medium was assayed to determine whether NAC could regulate glutamate level.Results:The spinal cord slices of normal control group had excellent organtypic cellular organization and their size became larger.There are about 24.13±3.44 motor neurons in the ventral horn in control group.The number of motor neurons in the ventral horn in THA group was markedly reduced compared with the control,only 6.27±1.62(P<0.05). Moreover,the glutamate concentration and LDH activity in the culture medium ,the MDA content in the spinal cord explants were significantly increased(P<0.05). Conversely,pretreatment with NAC significantly increased the number of motor neurons in the ventral horn ,19.29±2.13(P<0.05), and decreased the activity of LDH in the culture medium and the MDA content in the explants (P<0.05)compared with those in THA group. However, the glutamate concentration in the culture medium had no significant change(P>0.05).Conclusion:In the glutamate exitotoxicity spinal cord organotypic culture model,antioxidative agent NAC can protect motor neurons,and simultaneously alleviate the extent of cell injury and lipoperoxidation.However,NAC had no effect on the glutamate level in the culture medium.That is,the regulation of glutamate concentration in the culture medium of EGCG is not an additional result of antioxidation.In contrast,it is EGCG itself that regulate the glutamate level in the culture medium.更多还原