【作者】 吴璇；

【导师】 刘亚玲；

【作者基本信息】 河北医科大学 ， 神经病学， 2011， 硕士

【摘要】 目的:肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis ALS）是一种神经系统的变性疾病,一般认为ALS选择性损伤大脑皮层大锥体细胞及脑干运动神经核、脊髓前角的运动神经元,造成了脑和脊髓运动神经元的丢失,具体机制不明。然而在临床工作中发现ALS患者除运动系统症状外,部分伴有认知功能损害、感觉障碍、眼球运动障碍、自主神经功能障碍等运动系统外症状。因此,探讨ALS是否呈多系统累及,是我们需要关注的问题之一。ALS伴发的运动系统损害外症状中,以认知功能损害最为常见,而海马是公认的与认知功能有关的结构。在ALS患者的尸检研究中发现患者海马区可发现泛素阳性胞质内圆形、椭圆形包涵体,支持ALS累及非运动系统,并可能与临床上常见的认知功能损害有关。泛素系统（UPS）主要由泛素活化酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成。其广泛存在于真核生物中,降解80%以上的细胞内错误折叠蛋白或其他突变蛋白,是精细的特异性的蛋白质降解系统,是体内重要的细胞调控体系。研究认为泛素蛋白酶体降解通路失调可导致异常蛋白在细胞内积聚,并进一步引起细胞功能紊乱及变性。因此,泛素蛋白在海马区的表达异常可导致底物蛋白在细胞中异常聚集,形成胞质内包涵体,损害海马神经元细胞的正常功能,导致认知功能的损害。本实验应用家族型肌萎缩侧索硬化动物模型—SOD1^G93A转基因小鼠,通过对动物的行为学改变及海马区病理学变化的研究,探讨ALS是否存在认知功能的损害及认知功能的损害与泛素海马区异常表达的相关性,为临床研究提供理论基础。方法:1实验动物模型的建立由B6SJL2 BTg ( SOD12^G93A ) 1Gur /J半合子雄鼠及B6SJLF1 /J + / +雌鼠（购自美国Jackson Lab）配种,在恒温（ 22～27℃）、恒湿（ 40～50% ）、无特殊病原菌的动物房中繁殖、饲养SOD1^G93A转基因鼠,喂以灭菌水及颗粒型鼠类饲料。剪取小鼠尾部组织1mm,提取DNA,PCR技扩增,PCR产物经1.5%琼脂糖凝胶电泳后,,在长波紫外光下观察条带结果,鉴定SOD1（+）小鼠及SOD1（-）同窝对照小鼠。2动物分组Y型迷宫预选对电击反应较敏感,逃避反应迅速的60只小鼠(SOD1^G93A转基因清洁小鼠30只、同窝阴性对照小鼠30只)供实验用。随机分为60天组、90天组及120天组（onset3.5分）,每组各20只。3 SOD1^G93A转基因小鼠评分标准应用Vercelli et al的行为学评分系统于SOD1^G93A转基因小鼠100天每周对动物的全身状态进行评分,1～5分定义如下:5:健康,不伴任何瘫痪症状4:轻微步态不稳及后肢瘫痪症状3:明显瘫痪及步态不稳2:后肢完全性瘫痪,动物仅靠前肢爬行1:后肢完全性瘫痪,动物主要呈侧卧位,和/或翻转后不能翻身或体重下降超过初始体重的20%。4跳台试验试验分两天进行:第一天,训练前先将不同病程的SOD1^G93A转基因清洁小鼠放入反应箱内自由活动3min,熟悉环境。然后将动物置于铜栅上,接通铜栅电源,通以36V交流电。动物受到电击,观察记录动物5 min内第一次找到平台的时间（为学习反应时间）及5 min内跳下平台的次数（为学习错误次数）,学习反应时间及学习错误次数用以评价小鼠的学习功能。第二天,学习训练24h后,将动物置于平台上,接通铜栅电源,通以36V交流电。记录受测的动物5 min内第一次跳下平台的时间（为记忆潜伏时间）,和5 min内跳下平台的次数（为记忆错误次数）,记忆潜伏时间及记忆错误次数用以评价小鼠的记忆功能。实验过程中,动物有前肢跃起指向性找到平台的动作记录入学习反应时间,前肢指向性触及铜栅记录入记忆潜伏时间及学习、记忆错误次数。5组织病理学在动物麻醉状态下,4%多聚甲醛左心室快速灌注,留取脑组织。4%多聚甲醛固定24h后,于视神经水平脑横断面暴露海马,梯度酒精脱水,石蜡包埋,组织切片,免疫组织化学染色,光学显微镜下观察海马区泛素的表达情况。结果:SOD1^G93A转基因小鼠的记忆潜伏时间较同窝阴性对照小鼠延长,60天、90天及120天SOD1^G93A转基因小鼠的记忆潜伏时间分别为68.00±47.16s,55.20±92.99s,110.10±116.52s,同窝阴性对照小鼠的记忆潜伏时间分别为65.60±89.94s,158.00±88.31s,169.80±122.96s;同时SOD1^G93A转基因小鼠5min内记忆错误次数较同窝阴性对照小鼠增多,60天、90天及120天SOD1^G93A转基因小鼠的5min内记忆错误次数分别为3.40±2.84次,5.20±3.08次,1.80±1.32次,同窝阴性对照小鼠的记忆错误次数分别为3.30±2.16次,2.30±1.95次,2.00±2.75次。经t检验,各组SOD1^G93A转基因小鼠的记忆潜伏时间及记忆错误次数均较同窝阴性对照小鼠存在显著性改变（P<0.05）,而学习反应时间及5min内学习错误次数较同窝阴性对照小鼠无明显差异性变化（P>0.05）。而随SOD1^G93A转基因小鼠病程的延长,其记忆潜伏时间之间并无显著性差异（P>0.05）,而5min内记忆错误次数随病程延长存在统计学意义上的增多（P<0.05）。免疫组织化学染色显示在SOD1^G93A转基因小鼠海马的CA1、CA3及齿状回区均可发现泛素蛋白在细胞核周体的新月状或环形包涵体或粗颗粒状沉积,而在其同窝阴性对照的小鼠海马的CA1、CA3及齿状回区亦存在泛素的广泛表达,但表现为细颗粒状细胞质内的弥漫性广泛表达。并且SOD1^G93A转基因小鼠海马CA1、CA3及齿状回区的泛素阳性反应细胞比率均较同窝阴性对照小鼠显著性增多（P<0.05）。结论:SOD1^G93A转基因小鼠的记忆功能在发病早期较同窝阴性对照小鼠已出现有统计学意义的减退,且5min内记忆错误次数随病程延长而增多,而学习功能受损不明显。但SOD1^G93A转基因小鼠所表现出的记忆潜伏时间的缩短,与病程长短之间并无明显的相关性。同时,在SOD1^G93A转基因小鼠的病理免疫组织化学染色可见海马区神经元核周存在显著地泛素化蛋白异常聚集,可能与之所表现出的记忆功能的受损相关。更多还原

【Abstract】 Objective: Amyotrophic lateral sclerosis（ALS）is a degenerative disease of nervous system, which leads to the lose of motor neurons in brain and spinal cord, while the pathogenesis is unknown. Generally, we take ALS as a disease that involved pyramidal cells of cerebrum cortex, motor nucleus of brainstem and anterior horn motor neurons of spinal cord selectivity. However,during clinical work, we find some ALS patients have non-motor system symptoms, for example cognitive function impairment, sensory disturbance, ocular motility disorders, autonomic nerves function impairment and so on. Therefore, to explore whether ALS was multi-system involvement, is one of the issues need to focus.Among the non-motor system symptoms of ALS, cognitive function impairment is more common. Autopsy found round or ellipsoidal ubiquitin inclusion body in hippocampus neurons, which in support of ALS involving non-motor systems, and may be a common clinical cognitive impairment related.Ubiquitin-proteasome system （UPS） contains ubiquitin-activating enzyme, ubiquitin conjugating enzyme, ubiquitin-protein ligase and 26S proteasome. It exists in eukaryotic organism generally. More than 80% of misfolding proteins or other mutant proteins within the cells are degradated by it. Also it is subtile and specific protein degradated system and important controlling cell system. Studies suggest that disorders of ubiquitin-proteasome degradation pathway can lead to accumulation of abnormal proteins in cells, and further lead to cell dysfunction and degeneration. Therefore, the abnormal expression of ubiquitin protein in the hippocampus can lead to the substrate protein aggregated abnormally in neurons, and form cytoplasm inclusion, which injury the normal function of hippocampus neurons and lead to cognitive function impairment.In present study, we used familial amyotrophic lateral sclerosis model- SOD1^G93A transgenic mice and observed the changes of mouse’behaviors and hippocampus structure, to explored whether ALS exist cognitive function impairment or not. The study could provide a theoretical basis for clinical research.Methods:1 developed animal modelUsing B6SJL2 BTg (SOD12^G93A) 1Gur /J hemizygote males and B6SJLF1 /J + / + females（ Jackson Lab, America） to breed. Raising them in constant temperature（22～27℃）, constant humidity（40～50%）, depuratory housing, eating sterilizated water and granular pattern food of murids. Clipping tail of ^G93A transgenic mice about 1mm, extracting DNA, using PCR amplification, through 1.5% agarose gel electrophoresis, observing straps under long wave ultraviolet light, to identificate positive or negative mutants.2 grouped laboratory animalwe select 60 mouse that is sensitive to electric shock through Y labyrinth （positive mutants :30 mouse, negative mutants:30 mouse ） （+/–mutants: 30 females and 30 males） and divide them into three group randomly:60 days,90 days and 120 days （onset 3.5）. There are 20 mouse in each group.3 SOD1^G93A transgenic mice score standardFor weekly assessment of general condition starting at day 100 we used a behavioural score system based on the score developed by Vercelli etc. from 1 to 5 defined as follows:5: healthy without any symptoms of paralysis,4: slight signs of destabilized gait and paralysis of the hind limbs,3: obvious paralysis and destabilized gait,2: fully developed paralysis of the hind limbs, animals only crawl on the forelimbs,1: fully developed paralysis of the hind limbs, animals predominantly lie on the side and/or are not able to straighten up after turning them on the back or lost more than 20% of their starting weight.4 step down testThe step down test lasts two days. The first day: Putting the mice into the step down equipment to move freely about 3 minutes. Then take it on the copper grille and connect 36V alternating current. To observe in 5 minutes after electric shock, how long it takes to find the refuge area at the first time （as learning action time） and how manytimes it jumps down from refuge area （as learning errors frequency）. Take all above as learning training results.The second day:24 hours after learning training,put transgene mice on the refuge area and connect 36 V alternating current.Take notes in the 5 minutes ,how long it take to jump down from refuge area （if overtopping 5 minutes, record it as 5 minutes） and how many times it jumps down from refuge area （as memory errors frequency）.Take all above as memory training results.5 pathologyPour 4%paraformaldehyde into left ventricle fast when animals under anesthesia. Take the tissue of brains and fix them with 4% paraformaldehyde about 24 hours. Exposure hippocampus at optic nerve plane, dehydrate in gradient alcohol, then the tissues of brains were embedded in paraffin and sectioned at 5μm thickness. These sections were strained with anti-ubiquitin body to observe the forms of ubiquitin cytoplasm inclusion.Results: Comparing the memory action time of SOD1^G93A transgenic mice to negative controls , it shortens apparently.The memory action time of the 60 days ,90 days , and 120 days SOD1^G93A transgenic mouse are 68.00±47.16s, 55.20±92.99s, 110.10±116.52s. While the the memory action time of the negative controls are 65.60±89.94s, 158.00±88.31s, 169.80±122.96s.At the same time, the memory errors frequency in 5 minutes of SOD1^G93A transgenic mouse are 3.40±2.84 times, 5.20±3.08 times, 1.80±1.32 times, and the negative controls are 3.30±2.16 times, 2.30±1.95 times, 2.00±2.75 times. Through independent-samples T test, each group’s memory action time and memory errors frequency in 5 minutes have statistical significance comparing to the negative controls（P<0.05）. And the memory errors frequency in 5 minutes increases with duration prolonged of ALS（P<0.05）. While the learning action time and learning errors frequency in 5 minutes have no statistical significance（P>0.05）.But the memory action time has no correlation with duration of ALS（P>0.05）.The pathology finds crescent-or ring-shaped ubiquitin cytoplasm inclusions and granular cytoplasmic staining without a well-defined structure in CA1, CA3 and dentate gyrus of the SOD1^G93A transgenic mouse’hippocampus through immunohistochemistry. The most common type of dentate fascia inclusion had a crescent or ring shape. While generally in the negative controls , ubiquitin expresses in CA1,CA3 and dentate gyrus neurons cytoplasm, showed as tiny granular cytoplasmic staining. And the rate of positive neurons has statistical significance.Conclusions: Comparing to the negative controls, the spatial discrimination memory ability of SOD1^G93A transgenic mouse are injuried at prophase of onset. While learning ability injuring is not notablely. But the memory ability impairment is not complete correlation with duration of ALS. However, immunohistochemistry of SOD1^G93A transgenic mouse finds apparente polyubiquitinated protein aggregated abnormally in neurons cytoplasmic of hippocampus, which maybe correlation with the memory ability impairment.更多还原