【作者】 卜晖；

【导师】 李春岩；

【作者基本信息】 河北医科大学 ， 神经病学， 2007， 博士

【摘要】 肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis ALS）是一个致命的神经变性疾病。它的主要病理改变是选择性地损伤运动神经元（包括大脑皮层运动神经元、脑干运动神经核和脊髓前角运动神经元）。临床表现为缓慢进展的四肢无力,累及呼吸肌,多于发病3-5年内死亡。ALS可分为家族型肌萎缩侧索硬化（Familial Amyotrophic Lateral Sclerosis,FALS）和散发型肌萎缩侧索硬化（Sporadic Amyotrophic Lateral Sclerosis,SALS）,90%以上为散发型不足10%为家族型。其中FALS主要是由于编码Cu/Zn超氧化物歧化酶（SOD1）的基因突变引起,其突变位点在21号染色体长臂Cu/Zn SOD基因内,即21q22.1-22.2。与ALS有关的SOD1基因的突变大约有100余种。在这些突变中SOD酶的活性基本正常或升高,SOD基因敲除的小鼠则没有发病。这些发现提示SOD1相关的ALS不是因为功能缺失而引起,而是可能由一种获得性毒性引起。在运动神经元变性过程中牵涉到几个机制:兴奋毒性、免疫反应、线粒体功能障碍及自由基损伤、蛋白质异常聚积、蛋白酶体功能改变和凋亡。虽然这些通路对运动神经元损伤的启动和进展都起作用,但是这些通路之间的相互反应以及哪个起主要作用还不明了。这些机制不是互相排斥的而是可以被一个共同的反应所激活,这一共同的反应的协调者正是氧化应激。尽管确切机制尚不明了,但在导致运动神经元损伤的过程中,增加的氧化应激是出现较早且持久的事件。氧化应激不仅在ALS的发病中起了突出的作用,并且它通过活化其它各个通路,引起更新一轮的氧化应激,促进了疾病的进展。所以许多研究的焦点集中在氧化应激和自由基损伤。哺乳动物细胞内还原型谷胱甘肽（glutathione GSH）是细胞内含量最丰富、最有效的反应氧族（reactive oxygen species ROS）的直接清除剂。通过GSH与氧化型谷胱甘肽（GSSG）的转换来调节ROS水平,GSH减少则ROS产生增加并促进氧化损伤。另外GSH也是许多抗氧化酶的关键底物,这些酶解毒过氧化氢和脂质过氧化的产物。正如CHI.L和KE.Y等应用离体和在体的实验证实那样,GSH的缺失加强了运动神经元的变性。GSH的合成是一个ATP依赖的两步酶促反应。第一步由γ-谷氨酸半胱氨酸连接酶（γ- Glutamate Cysteine ligase GCL）催化完成,第二步由GSH合成酶（GSH synthetase）催化完成。前者是重要的限速酶。分别有GCLC和GCLM两个亚单位组成。随着年龄的老化,在大鼠中枢神经系统组织GCLM基因表达的下调,伴随着GCL酶活力的减低和GSH水平的降低。说明在与年龄相关的神经变性疾病中GCL及GSH发挥重要作用。由谷胱甘肽S一转移酶（Glutathione S-transferase GST）家族是一个分布广泛,包括近百种同工酶在内的多基因大家族。GSTs属Ⅱ相酶防御系统。具有多种生物学功能如催化包括亲电子试剂（electrophiles）、致癌物（carcinogens）和有基因毒性、细胞毒性的异生化合物（xenobiotics）与还原型谷胱甘肽（GSH）连接,使其转变为亲水的易排泄物质。GST在神经细胞氧化防御方面起重要作用。所以诱导内源性Ⅱ相酶,如GST已经被提议作为治疗PD的方法和癌症的化学预防。本课题根据谷氨酸的慢性兴奋毒性机制,应用谷氨酸转运体抑制剂苏-羟天冬氨酸（THA）,抑制星形胶质细胞对细胞外谷氨酸的转运,最终导致运动神经元死亡。制备成慢性选择性运动神经元损伤的脊髓器官型培养模型总之,慢性选择性运动神经元损伤的脊髓器官型培养模型为神经保护剂在人类运动神经元疾病的临床试验和临床前试验间提供了密切的联系。该模型已经成功地预言了力如唑（riluzole）和加巴喷丁（neurontin）有效的运动神经元保护作用,这两种药已经用于人类运动神经元病。故本课题利用该模型,以诱导内源性抗氧化剂为切入点寻求理想的治疗靶点。在许多神经变性疾病的病因涉及到神经细胞内ROS的堆积,细胞中和这些反应中间产物的能力很大程度上依赖一个顺式作用元件的活化,这个顺式作用元件被命名为抗氧化反应元件（antioxidant response element ARE）。它存在于抗氧化蛋白和解毒酶基因的5’端区域。在人类、大鼠、小鼠的多种组织中及原代培养的星形胶质细胞、神经元中都已证实,ARE驱动的靶基因包括GCLC、GLCM和GSTs等。所以,本课题应用ARE活化剂,观察其在该模型中对运动神经元的作用,并进一步探讨作用机制。论文共分四部分:第一部分证实ARE活化剂对运动神经元确实存在保护作用。第二部分探讨这种保护作用伴随着该模型中GSH水平和Ca²⁺水平的变化,而与P75NTR水平无关。第三部分进一步证实GSH合成的限速酶GCL在该模型中存在异常,随着ARE的保护作用,GCL的基因水平发生改变,说明GSH水平的改变是其上游合成酶变化引起。第四部分探讨了GSH相关酶:GST在该模型中及应用ARE活化剂后基因和蛋白水平的变化。总之,本课题证实,在慢性选择性运动神经元损伤的脊髓器官型培养模型中存在着GSH及其相关酶的异常,通过ARE活化剂能够改变它们的表达,这种改变伴随着对运动神经元的保护作用。第一部分在脊髓器官型培养模型中ARE活化剂对慢性选择性运动神经元损伤的保护作用目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,应用不同类型的ARE活化剂,不同剂量、不同方式进行干预,观察它是否能保护运动神经元免受慢性谷氨酸兴奋毒性损伤。并摸索出恰当的干预方法。为保护运动神经元寻找一个新的靶点。方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,在培养液中分别加入不同浓度CPDT、D3T和tBHQ（5μmol/L、15μmol/L、30μmol/L）,并且提前干预组（提前48小时）和与THA同时干预组,培养4周后,用神经元的特异性SMI-32免疫组化染色,对不同时点脊髓腹角α运动神经元进行记数,用透射电镜观察神经元超微结构变化,与对照组做比较。结果:正常对照组脊髓片形态完好,α运动神经元数目恒定,模型组α运动神经元数目减少。在提前干预的CPDT、D3T和tBHQ组15μmol/L、30μmol/L浓度,培养4周后α运动神经元数目较模型组运动神经元数目增多,与正常对照组相似。电镜下观察显示超微结构损伤也不明显,细胞器保持相对完好。而5μmol/L的CPDT、D3T和THA同时干预的各组,脊髓片损伤与模型组相似,α运动神经元明显减少。结论:利用ARE活化剂CPDT、D3T和tBHQ能够明显地保护运动神经元免受慢性谷氨酸兴奋毒损伤,为进一步研究ALS的发病机制及探讨神经保护治疗提供了有效的方法。第二部分ARE活化剂对慢性运动神经元损伤模型GSH、Ca²⁺及P75NTR的影响目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,观察GSH、Ca²⁺及P75NTR的变化。进一步应用ARE活化剂观察,在其对运动神经元的保护作用的同时对这些指标的影响。方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT、D3T。培养4周时取出。借助细胞内硫醇的分子探针（mCB）应用多功能酶标仪荧光法,检测脊髓组织细胞内还原型谷胱甘肽（GSH）浓度。借助Flow 3-AM(膜通透的细胞内Ca²⁺指示剂)应用流式细胞仪检测脊髓组织细胞内Ca²⁺相对浓度。提取组织匀浆蛋白,用免疫印迹检测脊髓组织P75NTR的蛋白水平。结果:GSH在48h组30μmol/L的CPDT明显较MS组增高,而15μmol/L的CPDT组略高。30CPDT96h组和15CPDT96h组的GSH水平均高于MS组,说明CPDT引起的GSH的升高是时间和剂量依赖的。在THA组存在GSH水平的降低,应用两个浓度的CPDT提前干预后可以阻止这种降低,并使GSH水平升高3-4倍。THA引起细胞内Ca²⁺水平升高,提前48小时应用CPDT和D3T后,纠正了细胞内Ca²⁺水平的升高。但THA组和CPDT提前组均无P75NTR蛋白的表达。结论: ARE活化剂CPDT、D3T对运动神经元的保护作用,伴随着提高细胞内GSH水平和降低细胞内Ca²⁺。该模型中不存在对P75NTR依赖的凋亡,也未发现CPDT对该途径的影响。第三部分慢性选择性运动神经元损伤模型中GCL的基因表达及ARE活化剂对其影响目的:验证在体外培养的脊髓薄片中,能否如在其它组织和器官中一样, GCLC、GCLM基因作为ARE的靶基因被上调。进一步验证慢性选择性运动神经元损伤模型中是否有该目的基因的异常,以及ARE活化剂能否阻止这种异常的改变。方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT。培养4周时取出。采用TRIZOL一步法从培养的脊髓组织中提取总RNA。应用RT-PCR法检测两个目的基因GCLC和GCLM以β-actin基因作为对照。结果:30μmol/L CPDT提前48h干预组GCLCmRNA表达明显高于正常对照组（MS组）,15μmol/L CPDT提前48h干预组与MS组比较略高于MS组,但无统计学意义。但在干预48小时各组GCLMmRNA的表达与MS在比较无统计学意义。在培养4周的模型中,两个浓度的CPDT提前干预48小时,然后再同时加入THA。GCLCmRNA的表达在两个浓度的CPDT提前组均明显高于MS组和THA组。MS组和THA组之间无差异。而GCLMmRNA的表达,仅THA组明显低于MS组,而15、30μmol/L CPDT提前干预对其无影响。结论: ARE活化剂CPDT可以升高体外培养的脊髓器官型模型中GCLC的基因水平,而对GCLM基因无影响。在THA造成的模型中存在GCLM基因表达的降低。与其下游的GSH的含量的改变相一致。第四部分应用ARE活化剂对慢性选择性运动神经元损伤模型中GSTM的影响目的:检测与GSH相关的酶, ARE活化剂的靶基因之一GSTM的蛋白和基因水平在该模型中的的表达。进一步验证,ARE活化剂在对运动神经元保护作用的同时是否伴随GSTM的变化。方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度的ARE活化剂:CPDT、tBHQ。继续培养48小时或继续培养3周后取出。提取组织总蛋白和总RNA。应用RT-PCR和Western blot分别检测各组组织中GSTM的基因和蛋白水平。结果:在ARE活化剂CPDT和tBHQ干预48小时可以刺激体外培养的脊髓片模型中GSTM蛋白水平的升高。在培养4周的模型中,CPDT干预后GSTM的蛋白和基因水平均比正常对照组增高。THA组也较正常对照组增高。结论:作为ARE的靶基因之一,GSTM可以在ARE活化剂的刺激下,在体外培养的脊髓片模型中表达升高。伴随着运动神经元的保护作用,这种升高可以持续4周。更多还原

【Abstract】 Amyotrophic lateral sclerosis （ALS） is a common progressive neurodegenerative disease in central nervous system. It is characterized by selective degeneration of motor neurons in brain ,brain stem and spinal cord, which clinical features are delayed onset, chronic progression, weakness of limbs’muscles. Most patients died of respiratory failure 3-5 years later. ALS can be divided into familial amyotrophic lateral sclerosis （FALS） and sporadic amyotrophic lateral sclerosis （SALS）. FALS is less than 10% of the total ALS patients, while SALS is more than 90%.FALS is mainly caused by the mutation of copper/zinc superoxide dismutase （Cu/Zn SOD） gene, whose site is at 21q22.1～22.2. More than 100 different mutations of the SOD1 gene have been linked to familial ALS. For many of these mutations, SOD1 enzyme activity is actually normal or elevated, and SOD1 knockout mice have no disease phenotype. These findings indicate that SOD1-associated ALS is not caused by a loss of function, but rather a toxic gain of function.Several mechanisms are implicated in the pathogenesis of motor neuron degeneration, including excitotoxicity, immune activation, mitochondrial dysfunction and oxidative stress, protein aggregation, altered proteosomal function and apoptosis. Although disturbances in each of these pathways may contribute to amplification or even initiation of motor neuron injury, the temporal relation of these pathways and their primacy in dictating disease onset and progression are unclear. These mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress. Increased oxidative stress appears to be an early and sustained event in association with motor neuron death in ALS, although the specific mechanism leading to oxidative damage on motor neurons remains to be defined. Oxidative stress has a prominent role in the initiation of ALS and is capable of activating pathways that elicit additional oxidative stress and propagate disease.GSH is the most abundant and effective scavenger against ROS directly in mammalian cells. In addition, GSH is also a key substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxide products. CHI.L和KE.Y show that depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo.The synthesis of GSH involves the actions of two ATP-dependent enzymes,γ-glutamylcysteine ligase （GCL） and GSH synthetase. GCL, the rate-controlling enzyme in the overall pathway, is a heterodimer composed of a catalytic （GCLC） and a modulatory （GCLM）.There is a down-regulation of GCLM gene expression in rat CNS tissue during aging, accompany by reduced activity of GCL and GSH level. This means, GCL and GSH play an important role in age-related neuron degeneration disease.Glutathione S-transferase （GST） is a multigene family of more than 100 isoenzymes that catalyze the conjugation of GSH to a variety of electrophilic compounds。Glutathione transferases （GSTs; EC, 2.5.1.18） are phase II enzymes of defense that catalyze the conjugation of reduced glutathione to a wide range of electrophiles, carcinogens and other xenobiotics with genotoxic and cytotoxic activities。GSTs play an important role in protecting neurons against oxidative stress damage. Many study suggest induction of endogenous phase II enzymes,e g,GST may be a strategy for PD and AD.Based on the chronic excitotoxicity pathogenesis, incubation of organotypic spinal cord cultures in presence of threo-hydroxyaspartate （THA）, the inhibitor of astrocyte glutamate transporter, causes death of motor neurons. We established the model of chronic motor neuron degeneration.Isummary, the organotypic spinal cord model of chronic motor neuron degeneration promises preclinical feasibility testing of potential neuroprotectants with enhanced relevance for clinical trials in human motor neuron disease. This model has already successfully predicted the efficacy of motor neuron protection by riluzole and neurontin, which are being used for human motor neuron disease. In this model, we want to study neuroprotective effects by inducing endogenous antioxidant agents.The intraneuronal accumulation of reactive oxygen species has been implicated in the pathogenesis of many neurodegenerative diseases .The ability of a cell to neutralize reactive intermediates is, in part, dependent on the activation of a cis-acting regulatory element termed the antioxidant response element （ARE）. The ARE is located in the 5’-flanking region of many genes essential for both detoxification and antioxidant proteins. In many tissues of human, rat, mice and primary culture neurons and astrocytes, there are many ARE-drived gene including DCLC, GCLM and GSTs.The organotypic spinal cord model of chronic motor neuron degeneration were used to study the effect of ARE activator on motor neuron cell death and mechanism. The first part: we show that ARE activator completely inhibit glutamate-induced motor neuron death in these explants. The second part: we show that ARE activator inhibits glutamate-induced intracellular Ca²⁺ rise and decrease of tissue glutathione, but don’t affect P75NTR pathway.The third part: the rate-controlling enzyme of GSH synthesis, GCL is abnormal in the model, but ARE activator correct the abnormal, which is corresponding to GSH alteration. The forth part: we investigate GSH related enzyme-GSTM protein and mRNA level in the model, ARE activator can change its level, which is accompanied by motor neuron protection.PartⅠARE activator protects motor neuron against glutamate excitotoxity-induced motor neuron death in organotypic spinal cord model of chronic motor neuron degeneration ,Objective: In organotypic spinal cord model of chronic motor neuron degeneration , to investigate if ARE activator could protect motor neurons against glutamate excitotoxicity.We use three ARE activator :CPDT ,D3T and tBHQ at different concentration,at different time points. To explore new neuroprotective treatment. Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT ,D3T and tBHQ（5μmol/L、15μmol/L、30μmol/L） were continuously added into the culture medium after cultured seven days. Spinal cord slices were treated with THA, a combination of THA and ARE activator , or treatment with the ARE activator without THA for 48 hours .Then we use monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with different group.Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventralα-motor neurons. THA could produce a slow loss ofα-motor neurons. The complete neuron protection was achieved when the explants were pretreated by ARE activator: CPDT, D3T, tBHQ for 48 hour prior to initiating the combination treatment. Whereas ARE activator was only able to offer very limited neuron protection against THA-induced motor neuron death when the two agents were always added together to the culture medium,and same as 5μmol/L ARE activator.Conclusions: It is possibility that ARE activator may block glutamate toxicity to protects motor neuron against glutamate excitotoxity-induced motor neuron death. This subject could provide an effective studying pathogenesis and neuroprotection of ALS.PartⅡTissue glutathione （GSH） contents, intracellular Ca2+ levels and P75NTR protein in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: To study if CPDT stimulates GSH content in rat spinal cord explants Proctention motor neuron survival in CPDT-treated spinal cord explants is accompanied by GSH, intracellular Ca2+ levels and P75NTR protein alteration.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT, D3T were continuously added into the culture medium after cultured seven days. GSH in tissue homogenates was derivatized by monochlorobimane, then the derivatives （GSH-monochlorobimane） were measured using a fluorescence plate reader. Measurement of intracellular Ca²⁺ level with 8μM Flou3-AM, then them were immediately analyzed by flow cytometry to determine fluorescence intensity.Tissue P75NTR protein was measured by Western blot.Results: After on week recover, the spinal cord tissues were treated with 15μM , 30μMCPDT for 48hours, then collected , measured ,GSH level increase only in 30CPDT group, but continue culture for 96 hours,GSH level was increase eather in 15CPDT group or in 30CPDT group.In the present experiment, the spinal cord tissues were first treated with CPDT at 15 and 30μM for 48 h before combined treatment of CPDT and THA for 3 weeks. We show that THA treatment might deplete tissue GSH. Indeed, 3-week THA treatment markedly reduced GSH level. Significantly, CPDT not only prevented THA-induced GSH depletion but actually elevated tissue GSH level 3-4 fold over the control. We found that CPDT fully prevented both intracellular Ca²⁺ rise.In the model, we could not find any specific P75NTR protein expression , but we could see it on one day old rat, which is control. After treatment with CPDT, there is no any specific protein expression at 75KD marker can be seen.Conclusions: CPDT could increase GSH level in spinal cord tissues ,which is time-depented and dose-depented.THA treatment reduced GSH level and CPDT not only prevented that but all so elevated GSH level 3-4 fold .CPDT,D3T fully prevented intracellular Ca²⁺ rise. CPDT have no effection on P75NTR pathway.PartⅢARE target gene-GCL expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: to investigate one of ARE target gene GCL （include two subunit GCLC and GCLM） expression in organotypic spinal cord model of chronic motor neuron degeneration. If their alteration are accompany by motor neuron protection and ARE activitor can prevented THA-induced alteration.Methods: Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GCLC, GCLMmRNA.Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.RT-PCR was used to measure the expression of two genes, including GCLCand GCLMalong withβ-actin gene as a control. The Trizol method was used to extract total RNA from the rat spinal cord explants.Results: At treat for 48hours group, only the rat spinal cord explants treated with 30μmol/LCPDT can increase GCLCmRNA expression, 15μmol/LCPDT could not affect it. On the other hand, two concentration CPDT treatment spinal cord explants show no alteration in GCLMmRNA expression at all.In the THA plus CPDT group,. In THA group, GCLMmRNA expression is lower than others. However, THA seems to have no effect on GCLC expression. Just as above, the combination treatment of THA with CPDT could not cause increase in GCLMmRNA expression. But this way caused a more significant increase in GCLCmRNA expression.Conclusions: Hight concentration CPDT （30μM） could cause increase expression of GCLC, but could not affect GCLM. However THA decrease GCLMmRNA expression, but can not affect GCLC.CPDT could increase GCLCmRNA last for 4 weeks, accompany by protection motor neuron.PartⅣGSTM protein and gene expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: to investigate one of ARE target gene GSTM protein and mRNA expression in organotypic spinal cord model of chronic motor neuron degeneration, If their alteration are accompany by motor neuron protection and ARE activator can prevented THA-induced alteration.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GSTM protein.Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.Prepare whole tissue extracts for Western Blot analysis. The Trizol method was used to extract total RNA from the rat spinal cord explants.Results: In lumber spinal cord explants treated with CPDT for 48 h shows CPDT and tBHQ CPDT at 15 and 30μM caused significant increase in expression of GSTM protein.In cultured for 4 weeks group, exposure of the explants to CPDT in the presence of THA lead to increased expression of GSTM in mRNA and protein .Interestingly, THA itself also seems to positively modulate some of the genes, GSTM.Conclusions: CPDT and tBHQ could stimulates ARE target genes in rat spinal cord explants. Increased motor neuron survival in CPDT-treated spinal cord explants is accompanied by activation of ARE targen gene GSTM expression and protein level.更多还原