肌萎缩侧索硬化的临床、电生理特点及致病机制研究

发布时间：2018-05-21 07:52

本文选题：肌萎缩侧索硬化 + 临床表现　；参考：《山东大学》2017年博士论文

【摘要】：肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)是成人运动神经元病最常见的类型,由Charcot于1869年首先描述。ALS与其他运动神经元病的区别是上运动神经元和下运动神经元均发生变性。ALS主要的神经病理特征包括脊髓前角及脑干运动神经元广泛损失、皮质脊髓束变性和初级运动皮质大锥体神经元变性及损失。ALS的患病率约为5-7/10万,大多数ALS病例为散发性(sALS),5-10%为家族性(fALS)。无论患者是散发性还是家族性,均会发生进行性肌肉无力和肌肉萎缩。进行性肌肉无力可始于上肢或下肢的近端或远端,并可累及所有肌肉,包括与呼吸、言语及吞咽有关的肌肉。多数患者在被诊断2-5年后,死于呼吸衰竭。在疾病早期,下运动神经元和上运动神经元受累的不同模式使ALS的诊断面临挑战。由于ALS最初症状与其他脊髓疾病、周围神经病变及神经综合征相似,因而诊断延迟是其主要问题之一。由于缺乏特异的疾病标志,ALS临床诊断标准的建立基于下运动神经元和上运动神经元病变的存在及分布,并且需要使用排除过程来确定诊断。实验室检查、神经影像和肌肉活检都可能有助于鉴别诊断和排除与ALS类似的疾病过程,而一旦其他疾病过程被排除,电生理评估是确立ALS诊断的关键。并且,电生理评价不仅在疾病诊断中有用,而且有助于监测疾病的进展和对干预的反应,有助于确定预后。因此,目前ALS诊断主要依靠临床表现与神经电生理检查相结合。目前被识别的引起ALS的基因突变超过20种,其中最常见的突变发生于SOD1、RNA结合蛋白编码基因TARDBP、FUS,以及最近所认识C9orf72基因。2011年,C9orf72基因非编码区六核苷酸GGGGCC的致病性重复扩增现象在ALS和额颞叶痴呆患者中被发现并报道。通过对9p21位点上风险单倍型的研究认识到C9orf72基因中GGGGCC扩增。应用重复引物聚合酶链反应的测序,最终认为至少30次的重复扩增与ALS有关。与非C9orf72 ALS病例相比,C9orf72相关性ALS有其临床特征,伴发额颞叶痴呆的比例明显增高,容易出现认知功能障碍和精神行为异常,延髓起病的比例增高,发病年龄提前1.8-5.0年,运动症状恶化速度快,病程缩短5.7-12.0月,显示了其更强的疾病进程。同时,C9orf72疾病谱还包括原发性侧索硬化、进行性肌萎缩、帕金森综合征/帕金森病、亨廷顿病、阿尔茨海默病等。由于携带C9orf72基因突变的患者数量众多,并且C9orf72相关疾病谱非常广泛,其介导的ALS是多方面且复杂的,近年来C9orf72基因逐渐成为研究热点。核因子κB(nuclear factor kappa B,NF-κB)是淋巴细胞中的一种诱导型转录因子,对身体中几乎所有细胞类型都产生影响,在炎症、免疫反应、细胞周期和细胞存活中起重要作用。在中枢神经系统,NF-κB转录因子是神经发生、神经突生成及突触可塑性等许多生理过程的关键参与者。NF-κB为组成型活性,参与神经元细胞体中的神经元损伤以及神经保护。NF-1κB在突触中以潜在形式存在,只有当其被激活时,才能被运输到神经元细胞核。免疫组化证实,NF-κB可在家族和散发ALS患者的神经胶质中被活化。研究表明,SOD1-G93A小胶质细胞在NF-κB依赖性机制中在体内及体外均可诱导运动神经元死亡。第一部分肌萎缩侧索硬化的临床及电生理特点分析目的:通过分析ALS临床及电生理资料,为临床医生提供本病较为直观和深入的认识,使患者尽可能得到早期诊断,从而获得正确的治疗,以期患者的生存期得以延长,生活质量得以提高。方法:收集2009年1月至2016年6月在我院神经内科就诊的73例患者,符合E1 Escorial修订版诊断标准,诊断分级为临床确诊及临床拟诊。进行详尽的病史询问及神经查体,收集患者的发病年龄、起病部位、累及部位、主要的症状及体征,进行运动传导速度(motor conduction velocity,MCV)测定、感觉传导速度(sensory conduction velocity,SCV)测定、重复神经刺激(repetitive nerve stimulation,RNS)、针电极肌电图(EMG)等电生理检查,回顾性分析、总结ALS的临床及电生理特点。结果:患者共73例,其中男42例(57.5%),女性31例(42.5%),男女比例为1.4:1。发病年龄28.1-78.8岁,平均发病年龄(51.7±12.4)岁。发病年龄高峰为51-60岁。起病部位比率最高的颈段为56.2%,其次为腰骶段(24.6%)和延髓段(19.2%)。常见的下运动神经元症状及体征中肌肉无力(97.3%)和肌肉萎缩(76.7%)最常见。运动神经传导总的异常率为45.9%,但运动传导的远端潜伏期、波幅及传导速度的平均值均在正常范围。复合肌肉动作电位(compound muscle action potential,CMAP)波幅值与截断值的比值,正中神经平均值为(1.06±0.74),尺神经平均值为(1.49±0.81),尺神经明显大于正中神经(P=0.000)。感觉神经传导总的异常率为2.3%,但感觉传导的波幅及传导速度的平均值均在正常范围。重复神经刺激,拇短展肌(正中神经支配)和小指展肌(尺神经支配)记录的CMAP波幅递减的平均值分别为(9.07%±8.68%)和(2.89%±2.47%),拇短展肌的递减幅度明显大于小指展肌(P=0.000)。在正中神经,CMAP递减和CMAP的波幅呈负相关(R=-0.357,P=0.000),而在尺神经,没有观察到相关性(P=0.223)。针电极EMG显示,舌肌总的自发电位及纤颤/正尖波、束颤电位的出现率(分别为 79.1%、65.1%、58.1%)均比胸锁乳突肌(分别为 48.8%、30.2%、32.6%)高(P值分别为0.004,0.001,0.017)。具有延髓症状和仅具肢体症状的患者,其舌肌自发电位的出现率分别为84.2%和75.0%,二者相比无统计学差异(P=0.708)。结论:ALS发病男性多于女性,好发于中老年人,发病高峰为51-60岁。起病部位以颈段最多,其次为腰骶段、延髓段。下运动神经元症状以肌肉无力最多见,其次为肌肉萎缩。部分患者会出现运动传导异常,以CMAP波幅下降最为多见。感觉传导异常少见,说明本病对感觉传导通路影响不大;如果出现异常,并不能完全排除本病,应注意排除伴随的其他周围神经病。RNS检查表明,正中神经比尺神经CMAP波幅递减的幅度大,其原因是在ALS的病理生理中正中神经较尺神经优先受累。CMAP递减可能由新芽生的神经末梢传导不稳定引起。在评估舌肌的临床和亚临床受累方面,无论是延髓起病还是肢体起病的ALS,舌肌针电极EMG均是一种有价值的电生理方法,但应注意操作方法。第二部分ALS突变基因C9orf72细胞模型的生物学功能分析及其机制的研究目的:构建C9orf72诱导的ALS细胞模型并对其进行生物学功能分析;检测C9orf72是否通过NF-κB信号通路造成神经元损伤。方法:利用化学合成方法,体外获得C9orf72的突变基因,然后构建基因的真核表达载体;应用阳离子脂质体转染法,将上述构建成功的真核表达载体转染到NSC-34细胞中,使用RT-PCR和Western blots方法,分别在转录组与蛋白质组进行检测,确认细胞模型构建成功;使用免疫细胞化学方法,进行神经元细胞的形态学检测;使用丙二醛(MDA)方法,检测细胞氧化应激情况;实验组与对照组,应用RT-PCR和Western blots方法,进行NF-κB信号通路活性检测;将siRNA-NF-κB转染到NSC-34细胞中,应用Western blots方法,研究NF-κB信号通路中,实验组与对照组的上下游蛋白分子变化,根据变化情况去阐明C9orf72诱导的ALS发生的分子机制。结果:获得转染 pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 成功的细胞模型。免疫细胞化学检测发现,与对照组相比,转染pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 后,胞体明显变圆,突起减少,且轴突变短。MDA结果显示,转染组为(2.92±0.14)nmol/mgprot,正常组为(1.63±0.12)nmol/mgprot,空载组为(1.82±0.21)nmol/mgprot,转染组细胞内MDA的含量明显高于其他两组(P0.05)。转染pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 24 小时后,以正常细胞组和转染pcDNA3.1(+)组作为对照组,与对照组相比较,转染组中细胞内的P-NF-κB蛋白表达明显升高(P0.05)。转染siRNA-NF-κB 24小时后,与siRNA转染组相比较,siRNA-NF-KB转染组中细胞内的NF-κB表达受到明显的抑制(P0.05);但是C9orf72蛋白的表达无变化。正常组、空载体组、pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 转染组与 siRNA-NF-κB转染组的 MDA 含量分别为(1.72±0.22)nmol/mgprot、(1.86±0.37)nmol/mgprot、(3.12±0.28)nmol/mgprot、(1.75±0.18)nmol/mgprot。统计学分析显示,pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 转染组细胞内的 MDA含量明显高于其他三组细胞(P0.05),而siRNA-NF-KB转染组的MDA含量与正常组和空载体组相比没有明显差异(P0.05)。结论:本研究成功构建了 C9orf72诱导的ALS细胞模型。C9orf72基因中30次重复的GGGGCC对神经元样细胞存在毒性损伤作用。C9orf72通过下游NF-κB信号通路导致神经元损伤。
[Abstract]:Amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) is the most common type of adult motor neuron disease. It was first described by Charcot in 1869 that the difference between.ALS and other motor neuron diseases was that both upper motor and lower motor neurons were the main degenerative.ALS of the degeneration.ALS including the anterior horn of the spinal cord and the deity of the brain stem. The prevalence of.ALS was about 5-7/10 million after extensive loss, corticospinal degeneration, and primary motor cortex major pyramidal neuron degeneration and loss. Most of the ALS cases were sporadic (sALS) and 5-10% was familial (fALS). Progressive muscle weakness and muscular atrophy were found in patients with sporadic or familial, progressive muscle weakness. It can begin at the proximal or distal end of the upper or lower limbs and can involve all muscles, including the muscles associated with respiration, speech, and swallowing. Most patients die of respiratory failure after 2-5 years of diagnosis. In the early stages of the disease, the different patterns of the involvement of the motor neurons and the upper motor neurons in the early stage of the disease challenge the diagnosis of ALS. Because of the initial symptoms of ALS, His spinal cord disease, peripheral neuropathy, and neurologic syndrome are similar, so the diagnosis delay is one of the major problems. Due to the lack of specific disease markers, the establishment of the ALS clinical diagnostic criteria is based on the presence and distribution of the lower motor neuron and upper motor neuron disease, and needs to use the exclusion process to determine the diagnosis. Laboratory examination, Neuroimaging and muscle biopsy may help identify and exclude the process of disease similar to ALS, and electrophysiological assessment is the key to the establishment of a ALS diagnosis once other disease processes are excluded. And electrophysiological evaluation is not only useful in the diagnosis of disease but also helps to monitor the progress of the disease and the response to intervention. Therefore, the current ALS diagnosis relies mainly on the combination of clinical and neuroelectrophysiological tests. The current identification of ALS gene mutations is more than 20, of which the most common mutations occur in SOD1, RNA binding protein encoding gene TARDBP, FUS, and the recent recognition of the C9orf72 gene.2011, and the non coding region six nucleosides of the C9orf72 gene. The pathogenicity repeat amplification of acid GGGGCC was found in ALS and patients with frontoleaf dementia. The GGGGCC amplification in the C9orf72 gene was recognized by the study of the risk haplotype at the 9p21 locus. The sequence of repeated primer polymerase chain reaction was used to conclude that at least 30 repeated amplification was associated with ALS. C9orf72 related ALS has its clinical features, a significant increase in the proportion of frontotemporal dementia, the incidence of cognitive dysfunction and abnormal mental behavior, the increase in the proportion of the onset of the medulla, the age of 1.8-5.0 in advance, the rapid deterioration of motor symptoms, and the duration of the disease for 5.7-12.0 months, showing a stronger disease process. At the same time, C9orf72 disease The disease also includes primary sclerosis, progressive myosclerosis, Parkinson's syndrome / Parkinson's disease, Huntington's disease, Alzheimer's disease, and so on. The number of patients with the C9orf72 gene mutation is numerous, and the spectrum of C9orf72 related diseases is very extensive. The ALS is multidimensional and complex. In recent years, the C9orf72 gene has gradually become a study. The nuclear factor kappa B (nuclear factor kappa B, NF- kappa B) is an inducible transcription factor in the lymphocyte, which affects almost all types of cells in the body and plays an important role in inflammation, immune response, cell cycle and cell survival. In the central nervous system, the NF- kappa B transcription factor is neurogenesis, neurite production and process. .NF- kappa B, a key participant in many physiological processes such as plasticity, is a constituent activity, participates in neuronal damage in the neuron cell body, and the neuroprotective.NF-1 kappa B exists in the synapse in a potential form only when it is activated can it be transported to the nucleus of the neuron. The immunization of NF- kappa B can be found in the family and in the emission of ALS patients. The study shows that SOD1-G93A microglia can induce the death of motoneurons in the NF- kappa B dependent mechanism in vivo and in vitro. The clinical and electrophysiological characteristics of the first part of amyotrophic lateral sclerosis (amyotrophic lateral sclerosis) are analyzed. By analyzing the clinical and electrophysiological data of ALS, it is more intuitive to provide this disease to clinicians. In order to get the patient to get the early diagnosis as far as possible so as to get the correct treatment so that the patient's life can be prolonged and the quality of life is improved. Methods: 73 patients in the neurology department of our hospital from January 2009 to June 2016 were collected, and the diagnostic criteria of E1 Escorial revision were conformed to the clinical diagnosis and clinical diagnosis. A detailed medical history inquiry and neural examination were carried out to collect the age of the patients, the location of the onset, the areas involved, the main symptoms and signs, the motor conduction velocity (MCV), the sensory conduction velocity (sensory conduction velocity, SCV), and the repetitive nerve stimulation (repetitive nerve stimulation, RN). S), electrophysiological examinations such as needle electrode electromyography (EMG), retrospective analysis, and summary of the clinical and electrophysiological characteristics of ALS. Results: there were 73 patients with 42 males (57.5%) and 31 women (42.5%). The male and female ratio was 28.1-78.8 years of age of 1.4:1., the average age of onset was (51.7 + 12.4) years. The peak of onset age was 51-60 years. The ratio of the onset of the disease was the highest. The cervical segment was 56.2%, followed by the lumbosacral segment (24.6%) and the medulla segment (19.2%). The most common symptoms and signs of the lower motor neurons were muscle weakness (97.3%) and muscle atrophy (76.7%). The total abnormal rate of motor nerve conduction was 45.9%, but the average value of the distal latency, amplitude and conduction velocity of the motor conduction was in the normal range. Complex muscle movement. The ratio of amplitude and truncation value of compound muscle action potential (CMAP), median nerve mean value (1.06 + 0.74), average of ulnar nerve (1.49 + 0.81), and ulnar nerve greater than median nerve (P=0.000). The total abnormal rate of sensory nerve conduction is 2.3%, but the average value of amplitude and conduction velocity of sensory conduction is normal. The average value of CMAP amplitude reduction recorded by repetitive nerve stimulation, abductor abductor muscle (median nerve innervation) and the small digital abductor (ulnar innervation) was (9.07% + 8.68%) and (2.89% + 2.47%), and the decrease of abductor pollicis abductor muscle was significantly greater than that of the small finger abductor muscle (P=0.000). In the median nerve, the decrease of CMAP and the amplitude of CMAP were negatively correlated (R=-0.357, P=0.00). 0) and in the ulnar nerve, no correlation was observed (P=0.223). The needle electrode EMG showed that the total spontaneous potential and fibrillation / positive tip of the tongue muscle were higher than the sternocleidomastoid (48.8%, 30.2%, 32.6%, respectively, 0.004,0.001,0.017, respectively, 65.1%, 65.1%, respectively), with the medullary symptoms and only limb symptoms. The incidence of spontaneous potential of tongue muscle was 84.2% and 75%, respectively, and there was no statistical difference between the two cases (P=0.708). Conclusion: the incidence of ALS is more than that of women. The onset of the onset is in the middle and old age, the peak of the onset is 51-60 years. The most common parts are the neck segment, the second is the lumbosacral segment, the medulla oblongata. Muscle atrophy. Some patients will have abnormal movement conduction with the most frequent decrease of CMAP amplitude. The abnormal sensation conduction is rare, which indicates that the disease has little influence on the sensory conduction pathway; if there is an abnormal occurrence and can not completely exclude the disease, it should be noted that the.RNS examination of the associated peripheral neuropathy should be excluded, and the amplitude of the median nerve CMAP wave should be excluded. The decrease is due to the fact that in the pathophysiology of ALS, the decreasing of the median nerve of the median nerve and the descending of the ulnar nerve may be caused by the instability of the nerve endings of the new buds. In assessing the clinical and subclinical involvement of the tongue muscles, both the medulla and the onset of the limb, the ALS of the lingual muscle needle electrode EMG is a valuable electricity. Physiological methods, but should pay attention to the operation method. Second part of the ALS mutant gene C9orf72 cell model biological function analysis and its mechanism research purpose: to construct the C9orf72 induced ALS cell model and to carry on the biological function analysis; detect whether C9orf72 can cause neuron damage through the NF- kappa B signal pathway. In addition, the mutant gene of C9orf72 was obtained in vitro, then the eukaryotic expression vector of the gene was constructed, and the eukaryotic expression vector was transfected into NSC-34 cells by the cationic liposome transfection method, and the RT-PCR and Western blots methods were used to detect the cells in the transcriptional group and the proteome respectively, confirming the success of the cell model construction. Using the immunocytochemical method, the morphological detection of neuron cells was carried out. The oxidative stress of cells was detected by using MDA method. The experimental group and the control group were used to detect the activity of NF- kappa B signaling pathway by using RT-PCR and Western blots methods; siRNA-NF- kappa B was transfected into NSC-34 cells. Western blots method was used to study NF-. In the kappa B signal pathway, the changes in the upper and lower protein molecules of the experimental group and the control group were changed to elucidate the molecular mechanism of the occurrence of C9orf72 induced ALS. Results: the successful transfection of pcDNA3.1 (+) - (CATCATCACCATCACCAT) (GGGGCC) 30 was a successful cell model. Immunological detection found that the transfection of pcDNA3.1 (+) - (CATCAT) to the control group was compared with the control group. CACCATCACCAT) after (GGGGCC) 30, the cell body became obviously round and the protuberance decreased, and the result of short.MDA showed that the transfection group was (2.92 + 0.14) nmol/mgprot, the normal group was (1.63 + 0.12) nmol/mgprot, and the empty group was (1.82 + 0.21) nmol/mgprot, and the content of MDA in the transfected group was significantly higher than that of the other two groups (P0.05). (GGGGCC) after 3024 hours, the normal cell group and the transfected pcDNA3.1 (+) group were used as the control group. Compared with the control group, the expression of P-NF- kappa B protein in the transfected group was significantly increased (P0.05). After transfection of siRNA-NF- kappa B, the expression of NF- kappa B expression in the cells in the siRNA-NF-KB transfected group was significantly inhibited (P0.0) was significantly inhibited (P0.0) (P0.0) was significantly inhibited (P0.0) in the transfected group (P0.0). 5) but there was no change in the expression of C9orf72 protein. The MDA content in the normal group, the empty body group, the pcDNA3.1 (+) - (GGGGCC) 30 transfection group and the siRNA-NF- kappa B transfection group were (1.72 + 0.22) nmol/mgprot, (1.86 + 0.37) nmol/mgprot, (3.12 + 0.28) nmol/mgprot, (1.75 + 0.18) nmol/mgprot. statistical analysis showed pcDNA3.1 (+) - (+) - (+) - (+) - (+) - (+) - (+) - (+) - (+) - (+) - (+) - ATCACCATCACCAT (GGGGCC) 30 transfected group was significantly higher than that of other three groups (P0.05), and the MDA content in siRNA-NF-KB transfected group was not significantly different from that of the normal group and the empty carrier group (P0.05). Conclusion: This study successfully constructed the GGGGCC pair of C9orf72 induced ALS cell model.C9orf72 gene for 30 repetitions in the C9orf72 induced.C9orf72 gene. The cytotoxicity of.C9orf72 cells was observed, and the damage of neurons was induced by the downstream NF- kappa B signaling pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R744.8

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