【作者】 王娜；

【导师】 卜晖；

【作者基本信息】 河北医科大学 ， 神经病学（专业学位）， 2013， 硕士

【摘要】 目的：肌萎缩侧索硬化症（ALS）是神经系统变性疾病的一种，属于运动神经元病的范畴，上下运动神经元均会受累，该疾病起病隐匿，发病前期常可以出现肢体疲乏、运动耐力下降等非特异性的症状，继而出现肢体远端无力并逐渐向肢体近端及躯干、头颈肌肉延伸，出现肢体瘫痪、肌肉萎缩，并最终因呼吸肌无力致呼吸衰竭或肺部感染而死亡。ALS的发病率约为1.5/10万，其中约10%的患者具有家族遗传性（fALS），另外大部分病例为散发性（sALS）。肌萎缩侧索硬化症的发病机制涉及多种因素的共同作用，具有运动神经元病的共同特点：病因不明、疗效不好、预后不良。通过对占该疾病少数比例的家族性肌萎缩侧索硬化（fALS）患者研究发现，fALS中包含多种基因突变，其中铜/锌过氧化物歧化酶1（SOD1）基因、TDP-43基因以及FUS/TLS基因突变已经被证实，可以对一部分ALS的发病做出解释。而其中SOD1的基因突变与ALS的相关性研究最为深入，ALS1是ALS病人中含有SOD1的基因突变的一种亚型，这类患者约占fALS的20%。SOD1基因位于人类21号染色体上，包含5个外显子和4个内含子，迄今发现的与ALS有关的SOD1突变已经超过100种，其上的5个外显子中均有突变位点被发现，主要突变形式为错意和缺失等点突变，这些突变基本以常染色体显性形式被遗传。基于以上SOD1基因突变与ALS的相关性，目前，人类已经成功将人SOD1基因转入到小鼠体内，培养了hSOD1G93A转基因小鼠，随着研究的进展，该动物模型逐渐成熟，已经受到国际上的广泛认可。关于ALS的发病机制有很多学说，如基因突变、氧化应激等学说，最近国内外研究提出了内质网应激理论。内质网存在于所有哺乳动物的细胞质中，它是一个封闭的、由内膜折叠而成的管道系统，因其显微结构呈网状，故称为内质网，内质网是细胞内最大的亚细胞器，承担细胞内多种重要的生理功能，同时也参与到其他细胞成分如线粒体、细胞核的病理过程中。目前对ALS病人及实验动物的研究已经初步证实内质网应激与ALS的发病机制相关。正常情况下内质网可以监控蛋白质的质量并参与细胞内的钙离子调节。当细胞受到内源性或外源性的打击时，蛋白质加工运输发生紊乱，可以产生大量未折叠或者错误折叠的蛋白质，这些蛋白质聚集在内质网中，即表现出未折叠蛋白反应（UPR）。当错误蛋白超出内质网的负荷时，可以激活内质网应激，适当的内质网应激可以起到细胞保护功能，但细胞中发生过度的内质网应激则可以引起细胞功能障碍，甚至细胞凋亡。内质网上有三种跨膜蛋白，分别为IRE1（inositol-requiring enzyme1）、ATF6（activating transcription factor6）和PERK（PKR-like ER kinase）。发生内质网应激时，这三种蛋白分别与内质网伴侣分子GRP78/BIP分离而被激活，继而分别介导三条通路。其中活化的IRE-1产生核酸内切酶活性，对其下游XBP1的前体mRNA进行剪接，编码剪接的XBP1（sXBP1）蛋白，而活化的ATF6被转移到高尔基体水解形成的活化的p50-ATF6。sXBP1及p50-ATF6可以进入细胞核，激活ERS反应元件（ER stressresponse element, ERSE）诱导内质网相关基因的表达，从而促进错误蛋白的正确折叠。PERK通路在内质网应激发生后，发生自身磷酸化，进而磷酸化转录起始因子eIF2α，使细胞中广泛的蛋白正常翻译下调，从而减轻内质网处理蛋白的负荷。本实验小组在前期的研究中发现，内质网应激发生时，三条通路上均有相应指标（sXBP1、p50-ATF6、p-eIF2α等）蛋白水平的改变，本实验旨通过对hSOD1G93A转基因小鼠中XBP1、sXBP1及ATF6等的mRNA进行实时定量RT-PCR测定，以期了解各内质网应激通路中相关指标的变化，并了解应激起始于mRNA水平，或起自蛋白水平。方法：第一部分:实验小鼠的获得及基因筛定选取半合子雄鼠B6SJL-Tg（SOD1-G93A）1Gur/J与B6SJLF1/J+/+的雌鼠进行配种，获得子代小鼠，用剪取少许小鼠尾尖的方法，获取子代小鼠的DNA，利用PCR反应进行突变基因的扩增，并采用琼脂糖凝胶电泳鉴定分析，分别得到含有hSOD1G93A突变基因的阳性小鼠及不含该突变基因的阴性小鼠。第二部分：hSOD1G93A转基因小鼠腰髓细胞中XBP1、sXBP1及ATF6的mRNA的变化研究1、分组：对鉴定后得到的hSOD1G93A转基因阳性的小鼠进行运动行为的观察及分组，分为出生28天组、35天组、49天组、63天组、症状早期（约90天）组及终末期（约120天）组，并分别取同期同窝基因鉴定为阴性的小鼠做对照。2、实验取材：按照各分组的时间，腹腔注射10%水合氯醛进行小鼠麻醉，然后快速取小鼠腰髓，迅速液氮冷冻，-80℃保存备用。3、实时定量RT-PCR：采用Trizol试剂法提取小鼠腰髓总RNA，测取RNA浓度，以RNA为模板反转录为cDNA，继而以cDNA为模板利用XBP1、sXBP1及ATF6的特异性引物，应用实时定量荧光PCR的方法分别进行扩增。观察以上各指标的mRNA在各组小鼠腰髓中的含量变化。结果：实时定量RT-PCR结果显示: XBP1、sXBP1的mRNA在症状早期和终末期的转基因小鼠组与同期对照组相比表达增高，症状早期增高更为明显。而ATF6的mRNA在各转基因组与对照组之间无明显差异，且在不同时期转基因组之间和不同时期对照组之间均未见明显差异。结论：本实验通过对hSOD1G93A转基因小鼠不同时期内质网应激标志物sXBP1、XBP1及ATF6的mRNA进行实时定量RT-PCR研究发现：hSOD1G93A转基因小鼠不同时期内质网应激程度不同，症状早期及终末期sXBP1、XBP1的mRNA比同期对照组增多，症状早期最为显著。而ATF6的mRNA在各期均未见明显变化。更多还原

【Abstract】 Amyotrophic lateral sclerosis (ALS) is a kind of neurologicaldegenerative disease. It belongs to motor neuron disease, both upper andlower motor neurons are involved. Before the disease, patients often appearsome nonspecific symptoms. For example: body fatigue, poor-sportsendurance and so on. The limitation about the activities in unilateral orbilateral distal end of limbs and movement weekness appear as the onsetsymptom. The muscles of proximal end of the limbs, trunk and neck areweekness, paralysis and atrophy, finally the patient ofen die in respiratoryfailure or lung infection caused by respiratory muscles atrophy and weekness.The incidence of ALS is1to2per100,000, person. Approximately10%ofALS patients are inherited (fALS), and most cases of ALS are sporadic(sALS). There is most common characteristic of motor neuron disease inamyotrophic lateral sclerosis, unknown etiology, poor curative effect andunfavourable prognosis, the pathogenesis of ALS involves a variety of factors.Several studies about fALS show that a variety of gene mutation residedin fALS, zinc-dependent superoxide dismutase1(SOD1) gene, TDP-43gene,FUS/TLS gene and so on. The greatest contribution to ALS come from thediscovery of mutations in the SOD1, ALS1is a subtype of ALS with SOD1Mutations.That can account for20%of familial ALS. SOD1gene locates onchromosome21, with five exon and four introns. More than100SOD1genemutations about ALS have been reported. These mutations existed widely inall the five exons. Mostly mutations are missense and deletion. Therefor,human SOD1gene was transduced into mice successfully. This hSOD1(hSOD1) mutations transgenic mice used widely.There was a lot of theory about the pathogenesis of ALS, for example: genetic mutation, oxidative stress and so on. Recently，several studies haveshown that endoplasmic reticulum stress play an important role in ALS.Endoplasmic reticulum is an organelle in the cytoplasm of all mammals. It is acomplicate, meshy closed piping system, undertaked important physiologicalfunction in cells. Endoplasmic reticulum involved into the pathologicalprocess of nucleolus and mitochondria. The correlation of endoplasmicreticulum stress and ALS has been confirmed both in experimentation onanimals and ALS patients.Normally, endoplasmic reticulum was the monitor of the quality of mostprotein and acted as an important intracellular calcium store. Whenendogenous and extrinsic stress factors encroached on cell, the processing ofprotein is disturbed and a large number of unfolded protein was produced.Then, over-accumulated unfold proteins resulted in unfolded protein response(UPR). The abnormal protein was cleared by the way of ER associateddegradation (ERAD). Appropriate ERS play important role in protecting cells,however, excessive ERS cause Cell-dysfunction, leading to neuron apoptosisultimately.There are three kinds of transmembrane protein in Endoplasmicreticulum.they are IRE1(inositol-requiring enzyme1)、ATF6(activatingtranscription factor6) and PERK (PKR-like ER kinase), GRP78/BIP andthere transmembrane protein are dissociated with the activation of therepathway. The activation of the IRE1can splice XBP1mRNA, encode splicedXBP1(sXBP1) protein. The activation of the ATF6was transferred into Golgibody and cut to activited p50-ATF6, sXBP1and p50ATF6could translocatesto the nucleus, Combined with ER stress response element (ERSE) andincrease the expression of some ERS related protein. PERK phosphorylateseukaryotic initiation factor2α (eIF2α), which results in the decreasedtranslation of lots of protein to protecting cell from damage.This hSOD1mutation transgenic mouse is most widely usedexperimental model. Endoplasmic reticulum stress existed in the hSOD1G93Amice. This study is to find the change of the mRNA of XBP1, sXBP1and p50 ATF6in the hSOD1G93Amice to find the relation between ERS and ALS.Methods: Part one: hSOD1G93Atransgenic mice and identifyHemizygous males B6SJL-Tg (SOD1-G93A)1Gur/J and femalesB6SJLF1/J+/+breeding, Obtaining progeny mice, shearing tip of its tail,extracting DNA, PCR amplification, and the PCR product waselectrophoresed on agarose gel analysis. After identifed, we can obtainhSOD1G93Atransgenic mice, and wtSOD1mice without hSOD1.Part two:To research the change of mRNA about XBP1、sXBP1and p50ATF6in the spinal cells of hSOD1G93Atransgenic mice.(1)Experimental group：28days old hSOD1G93Atransgenic mice,35daysold hSOD1G93Atransgenic mice,49days old hSOD1G93Atransgenic mice,63days old hSOD1G93Atransgenic mice, symptoms of hSOD1G93Atransgenic mice3mice (about90days), end-stage hSOD1G93Atransgenicmice (about120days). Every group chooses wtSOD1mice from same broodas control.(2)Drawn materials: at each points, anesthetized mouse with10percent chloral hydrate, remove the waist pulp of mouse, liquid nitrogenfrozen swiftly. Then specimens was transfered into-80℃.(3)Real-time RT-PCR: extract the total RNA with Trizol reagent,measured the concentration of total RNA, reversed transcription to cDNA,real time RT-PCR With cDNA templates using SYBRGreen, observed eachindicator in each group.Results: Real-time RT-PCR analyses demonstrated that the expressionsof mRNA of sXBP1and XBP1were different between controls and transgenemice at different stages. The levels of sXBP1and XBP1mRNA were moreabundant in on-set stage and end-stage transgenic mice than in age-matchedcontrols, on-set stage transgenic mice more symptomatic. Well, at every stage,the levels of ATF6mRNA did not differ between transgenic mice and controls.Conclusions: In this experiment we try to study in different stage ofhSOD1G93Atransgenic mice, the level of sXBP1and XBP1mRNA showsdiffer, it demonstrated the strength of ER stress showed difference in different stage. In on-set stage transgenic mice the sXBP1and XBP1mRNA was mostabundant, In this stage, ER stress is intense.更多还原