鞘内注射ssAAV9-DAO对ALS转基因小鼠模型的保护作用及机制研究

发布时间：2017-12-27 20:05

本文关键词：鞘内注射ssAAV9-DAO对ALS转基因小鼠模型的保护作用及机制研究　出处：《河北医科大学》2017年博士论文　论文类型：学位论文

【摘要】：肌萎缩侧索硬化(Amyotrophic lateral sclerosis,ALS)是一种成人起病的、进行性、致死性神经系统变性疾病,病变累及皮层、脑干及脊髓前角运动神经元,导致全身肌肉的进行性萎缩、无力。患者多于发病后的3~5年内因呼吸肌萎缩无力而死亡。由于其发病机制的复杂性,目前尚无有效的治疗方法。ALS的患病率约为5~7/10万,其中约90%为散发型肌萎缩侧索硬化(SALS),其余10%为家族性肌萎缩侧索硬化(FALS),约20%的FALS是由铜/锌超氧化物歧化酶1基因(SOD1)的突变引起。SOD1-G93A转基因小鼠为SOD1基因93号位点发生甘氨酸→精氨酸的突变,高度模拟人类ALS疾病的病理及临床特点,被广泛地应用于ALS病理机制的研究。ALS的病理机制目前尚未完全明确,已知的有蛋白错误折叠、兴奋性氨基酸毒性、线粒体损伤、氧化应激等,近年来,一些新的证据表明D-丝氨酸(D-serin,D-Ser)—一种内源性的N-甲基-D-天冬氨酸受体(NMDAR)的协同激动剂,在ALS病人中有所升高,并可加剧运动神经元的死亡。而造成D-Ser升高的主要原因在于D-氨基酸氧化酶(D-amino acid oxidase DAO)的活性降低。DAO是一种以黄素腺嘌呤二核苷酸(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸如D-丙氨酸、D-丝氨酸、D-脯氨酸的氨基生成相应的酮酸。DAO广泛存在于哺乳动物的中枢神经系统(CNS)、肝脏及肾脏,在CNS主要存在于脑干和脊髓。在哺乳动物和低等生物如酵母、真菌和细菌的DAO蛋白之中,Arg199残基紧靠FAD结合位点,处于Tyr228和His307残基之间,对酶活性起着至关重要的作用。发生于此位点的突变(R199W)会在细胞中引起不良后果,可导致ALS的发生发展。研究认为,脊髓内存在的高水平的DAO主要作用在于降解D-Ser,从而通过NMDAR介导的神经毒性调控神经元的存活。而不论在f ALS患者中还是在SOD1-G93A模型小鼠中,腰髓DAO水平均有所降低。我们设想补充ALS小鼠模型腰髓中的DAO水平可能会增加D-Ser的降解,从而减轻NMDAR过度激活造成的神经毒性,保护运动神经元。自1952年DNA被发现作为遗传物质以来,利用载体(如病毒)携带DNA以治疗遗传性疾病的方法便应运而生。常用于转导中枢神经系统的载体有包含质粒的纳米颗粒、逆转录病毒重组载体、腺相关病毒(AAV)、腺病毒和单纯疱疹病毒(HSV)。其中AAV已成为最安全、最常用的一种用于传递治疗基因的病毒载体。本研究应用CMV启动子调控下的单链腺相关病毒血清9型(ss AAV9),携带编码的小鼠DAO基因(ss AAV9-DAO)经鞘内注射的途径干预发病早期的SOD1-G93A转基因小鼠,观察上调小鼠腰髓内的DAO的水平对其行为学及病理表现的影响,并进一步探索其可能的机制。我们查阅文献尚未见类似报道。第一部分ALS小鼠模型中DAO的表达及分布目的:我们对不同时期(60天龄—症状前期、90天龄—症状早期、120~140天龄—终末期)的SOD1-G93A转基因小鼠腰髓组织内的DAO水平进行检测,观察DAO水平随病程进展发生的变化,并进一步观察DAO所在的细胞类型,为病毒载体系统的选择提供依据。方法:我们选取由美国Jackson实验室提供的SOD1-G93A转基因小鼠作为动物模型,并应用其提供的引物序列和鉴定方法进行DNA鉴定,鉴定阳性的60天、90天及终末期小鼠作为研究对象,并以相同天龄的阴性小鼠作为阴性对照(即60天阳性、60天阴性、90天阳性、90天阴性、终末阳性、终末阴性共6组),每组4只小鼠。小鼠新鲜取材后采用蛋白免疫印迹法(Western blot)检测小鼠腰髓DAO的表达水平。另取90d阳性小鼠3只,灌注取材后行25μm震荡切片,采用免疫荧光共聚焦技术(confocal)观察DAO主要存在的细胞类型。结果:1与阴性鼠比较,SOD1-G93A小鼠腰髓DAO蛋白水平在终末期显著减少(P0.05),其他时期则无显著变化。2免疫荧光共聚焦染色结果显示SOD1-G93A小鼠腰髓DAO主要表达在运动神经元,此外星形胶质细胞和小胶质细胞也有极少量的表达。结论:SOD1-G93A小鼠腰髓组织内的DAO表达在终末期时显著减少,说明了补充腰髓DAO可能对SOD1-G93A小鼠具有保护作用;SOD1-G93A小鼠腰髓中DAO主要存在于运动神经元,这对选取神经元靶向的载体系统提供了依据。第二部分鞘内注射ss AAV9在SOD1-G93A转基因小鼠脊髓转导效率的研究目的:通过给SOD1-G93A转基因小鼠鞘内注射ss AAV9-GFP和ss AAV9-DAO,观察在此种方法下,绿色荧光蛋白(GFP)在SOD1-G93A小鼠脊髓内的分布情况及转导效率,以及小鼠腰髓组织DAO及D-Ser表达的变化,以确定此种方法的可行性。方法:选取SOD1-G93A转基因小鼠作为研究对象,单次鞘内注射ss AAV9-GFP(1×1012vg/kg)或ss AAV9-DAO(1×1012vg/kg),给药3周后部分小鼠以4%的多聚甲醛液灌注固定,行脊髓组织25μm震荡切片,部分小鼠新鲜取材,分离腰髓组织。通过免疫荧光共聚焦技术观察GFP表达及分布情况,蛋白免疫印迹法(Western blot)检测小鼠腰髓DAO的表达水平,免疫组织化学染色法观察小鼠腰髓D-Ser的表达。结果:1脊髓切片免疫荧光染色显示腰髓可见较多GFP表达,另外骶髓、胸髓也有少许GFP表达,颈髓则很少见。2免疫荧光共聚焦染色显示GFP与神经元共定位,而不表达于星形胶质细胞和小胶质细胞,且SOD1-G93A转基因小鼠腰髓运动神经元转导效率约为12%。3 Western blotting结果显示ss AAV9-DAO组小鼠腰髓组织DAO表达明显增多,同时免疫组织化学染色结果显示,与ss AAV9-GFP组小鼠相比,ss AAV9-DAO组小鼠腰髓组织D-Ser阳性细胞数显著减少。结论:GFP主要表达于注射点附近的脊髓部位,腰髓为主;GFP主要表达于神经元,且腰髓运动神经元转导效率约为12%;ss AAV9-DAO单次鞘内注射可增加SOD1-G93A小鼠腰髓内的DAO表达,同时降低D-Ser水平。说明了此种给药方式可补充小鼠腰髓内的DAO水平并具有酶活性。第三部分鞘内注射ss AAV9-DAO对SOD1-G93A转基因小鼠行为学影响及机制研究目的:通过给SOD1-G93A转基因小鼠鞘内注射ss AAV9-DAO,观察过表达DAO对SOD1-G93A小鼠生存期、体重、转轮试验及组织病理改变的影响,并探索相关的保护机制。方法:选取由美国Jackson实验室提供的SOD1-G93A转基因小鼠作为ALS动物模型,并应用其提供的引物序列和鉴定方法进行DNA鉴定,选择同窝配对的阳性鼠作为受试对象。小鼠90天龄时,选取体重接近(组间平均起始体重差别≤0.3g)的同窝小鼠,雌、雄各15对,随机分配到治疗组和对照组,分别给予ss AAV9-DAO和ss AAV9-GFP鞘内注射,观察小鼠体重、转轮变化及生存期。另外选取给予同样干预方法的小鼠进行机制研究,给药3周后取材,其中3对用0.05%的戊二醛-多聚甲醛液灌注固定,腰髓25μm震荡切片,通过免疫组织化学染色法观察SMI-32、Iba-1、GFAP的改变;L5前根切片甲苯胺蓝染色观察轴索破坏情况;余4对新鲜取材,腓肠肌冰冻切片HE染色观察肌纤维情况;通过蛋白免疫印迹法检测腰髓TNF-α、IKBα、IKKβ、IKKγ、p-P65、p-Akt、Bcl-2、Bax、Caspase3、Caspase9、Cd11b、GFAP的水平。结果:1与ss AAV9-GFP组小鼠相比,ss AAV9-DAO组小鼠生存期延长6天,差异有统计学意义(n=30,P0.05),其中,雌鼠延长8天,差异显著(n=15,P0.05),雄鼠也延长8天,但差异无统计学意义(n=15,P0.05)。雄鼠两组间的体重、转轮均无显著差异;雌鼠ss AAV9-DAO组仅在110天龄时短暂改善转轮表现(P0.05),体重则无显著差异。2免疫组织化学染色结果显示,与ss AAV9-GFP组小鼠相比,ss AA V9-DAO组小鼠腰髓组织SMI32表达增多、GFAP、Iba-1表达减少,同时Western blotting结果显示,ss AAV9-DAO组小鼠腰髓组织Cd11b、GFAP表达较对照组减少。即治疗组腰髓运动神经元保留较对照组明显增多,而星形胶质细胞、小胶质细胞增生较对照组明显减少。3 L5前根切片甲苯胺蓝染色结果显示,与对照组小鼠相比,给药组小鼠L5前根保留的轴索数量明显增多。腓肠肌冰冻切片HE染色结果显示,对照组小鼠肌纤维大小不一,可见大量中央核,给药组较之有所改善。4腰髓组织Western blotting结果显示,与ss AAV9-GFP组相比,ss AAV9-DAO组的NF-κB信号通路正向调节因子IKKβ、IKKγ水平显著降低,而负向调节因子IKBα显著增高,另外反映NF-κB信号通路激活状态的p-P65、TNF-α水平均显著降低(P0.05)。同时,ss AAV9-DAO组促凋亡因子Bax、Caspase3、Caspase9均显著降低,而抗凋亡因子Bcl-2显著增高,且伴有p-Akt表达的增高(P0.05)。结论:ss AAV9-DAO单次鞘内注射可延长症状期SOD1-G93A小鼠的生存期,但存在性别差异,造成此种差异的原因尚不完全明确。但对体重、转轮表现的改善不显著;ss AAV9-DAO可减少SOD1-G93A小鼠腰髓运动神经元的丢失和小胶质细胞、星形胶质细胞的增生活化,并对前根的轴索及肌肉纤维也具有一定保护作用;DAO的保护作用可能与其抑制NF-κB的激活从而抑制炎症反应,抑制Akt的失磷酸化从而减少凋亡反应有关。确切的机制尚有待进一步研究。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is an adult onset, progressive and fatal neurodegenerative disease. Lesions involve motor neurons in the cortex, brain stem and spinal cord, resulting in progressive atrophy and weakness of the whole muscle. The patients died of respiratory muscle atrophy in 3~5 years after the onset of the disease. Because of the complexity of its pathogenesis, there is no effective treatment at present. The prevalence of ALS is about 5~7/10 million, about 90% of which are sporadic amyotrophic lateral sclerosis (SALS), and the rest 10% are familial amyotrophic lateral sclerosis (FALS). About 20% of FALS is caused by mutations in Cu / Zn superoxide dismutase 1 gene (SOD1). SOD1-G93A transgenic mice have been genetically modified by glycine to arginine at site 93 of SOD1 gene, which is highly simulating the pathological and clinical characteristics of human ALS disease. It is widely used in ALS pathological mechanism research. The pathogenesis of ALS is not yet entirely clear, known to have protein misfolding, excitotoxicity, mitochondrial damage, oxidative stress, in recent years, some new evidence that D- serine (D-serin, D-Ser) - N- methyl -D- aspartate receptor (NMDAR) is a kind of endogenous CO agonist, has increased in ALS patients, and can aggravate the death of motor neurons. The main reason for the increase of D-Ser is that the activity of D- amino acid oxidase (D-amino acid oxidase DAO) is reduced. DAO is a typical flavin protease that uses flavin adenine dinucleotide (FAD) as a complementary base, and it can oxidize amino acids such as D- alanine, D- serine and D- proline to produce corresponding keto acids. D- can also be used to produce ketoacids. DAO exists widely in the central nervous system (CNS), liver and kidney of mammals. In CNS, it mainly exists in the brain stem and spinal cord. In mammalian and lower organisms, such as yeast, fungi and bacteria, the Arg199 residues are closely related to FAD binding sites, which are between Tyr228 and His307 residues, and play an important role in DAO activity. The mutation (R199W) that occurs at this site can cause adverse effects in the cell, which can lead to the development of ALS. It is believed that the major role of the high level of DAO in the spinal cord is to degrade D-Ser, which regulates the survival of neurons through NMDAR mediated neurotoxicity. Both in the f ALS patients and in the SOD1-G93A model mice, the DAO level of the lumbar spinal cord decreased. We assume that the DAO level in the lumbar spinal cord of the supplementary ALS mouse model may increase the degradation of D-Ser, thereby reducing the neurotoxicity caused by excessive activation of NMDAR and protecting the motor neurons. Since the discovery of DNA as a genetic material in 1952, the use of carriers (such as viruses) to carry DNA for the treatment of hereditary diseases has emerged. Vectors commonly used to transduce the central nervous system include nanoparticles containing plasmid, retroviral recombinant vectors, adeno-associated virus (AAV), adenovirus and herpes simplex virus (HSV). AAV has become the safest and most commonly used virus carrier for the transmission of therapeutic genes. The research and application of CMV promoter single stranded adeno-associated virus type 9 serum sub regulation (SS AAV9), mice carrying the DAO gene encoding (SS AAV9-DAO) by intrathecal injection of the interventions in the early onset of SOD1-G93A transgenic mice, to observe the effect of up regulation of spinal cord of mice in the DAO level of the behavior and pathological findings, and to explore its possible mechanism. We have not found similar reports in the literature. The first part of the expression and distribution of ALS in a mouse model of DAO Objective: we in different periods (60 days of age and symptoms early, at the age of 90 days and 120~140 days of age, early symptoms and ESRD) were detected in SOD1-G93A transgenic mouse spinal cord in the DAO level, to observe the change of DAO level with the development of course of disease occurrence. And to observe DAO's cell types, provide the basis for the selection of virus vector system. Methods: We selected SOD1-G93A transgenic mice provided by the United States Jackson laboratory as an animal model, and use of the primer sequences and identification methods of DNA identification, identification of positive 60 days, 90 days and end-stage mice as the research object, and on the same day old mice were used as negative control (i.e., positive for 60 days 60 days, 90 days, negative positive negative and positive at the end of 90 days, a total of 6 terminal negative group), 4 mice in each group. Western blot was used to detect the expression level of DAO in the lumbar spinal cord of mice. In addition, 3 90d positive mice were taken, and 25 mu m was sliced after perfusion. The main cell types of DAO were observed by immunofluorescence confocal technique (confocal). Results: 1 compared with the negative mice, the level of DAO protein in the lumbar spinal cord of SOD1-G93A mice decreased significantly at the end stage (P0.05), but there was no significant change at other times. 2 immunofluorescence confocal staining showed that DAO in lumbar spinal cord of SOD1-G93A mice was mainly expressed in motoneurons, and astrocytes and microglia also had very few expression. Conclusion: SOD1-G93A mice spinal cord tissue expression of DAO decreased significantly in end-stage, that of lumbar spinal DAO may have protective effects on SOD1-G93A mice; SOD1-G93A mice spinal cord in DAO are mainly in the selection of motor neurons, neuron targeting carrier system provides a basis for. The second part of intrathecal injection of SS AAV9 in the study of SOD1-G93A transgenic mouse spinal cord transduction efficiency to SOD1-G93A transgenic mice of intrathecal injection of SS AAV9-GFP and SS AAV9-DAO, were observed in this way, the green fluorescent protein (GFP) distribution and transduction efficiency in SOD1-G93A mouse spinal cord, and the expression of DAO and D-Ser in mice spinal cord tissue, to determine the feasibility of this method. Methods: SOD1-G93A transgenic mice were selected as subjects. Single dose intrathecal injection of SS AAV9-GFP (1 x 1012vg/kg) or SS AAV9-DAO (1 x 1012vg/kg) was administered for 3 weeks, and some mice were 4% polypeptides.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R744.8;R-332

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