ET-1及Notch1通路相关蛋白在肌萎缩侧索硬化转基因小鼠脊髓中的表达研究

发布时间：2018-03-21 12:48

本文选题：Notch1信号通路　切入点：少突胶质前体细胞　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)是一种累及皮层、脑干、皮质脊髓束以及脊髓运动神经元的神经变性疾病,主要的临床表现为进行性肌肉萎缩,导致运动功能严重障碍甚至呼吸肌麻痹,患者生存期一般为3-5年。据临床统计其发病率为1.5-2.5/10万。5%-10%为家族性ALS,其余90%以上属于散发型ALS。其发病机理涉及炎症、氧化应激、蛋白聚集、线粒体功能障碍、轴浆运输受损、谷氨酸兴奋性毒和神经营养因子不足等,任何单一假说都无法很好的解释ALS的发病机制。Notch信号通路在决定细胞结局、迁移分化、生长以及突触的塑造和神经元存活中扮演重要的角色。Notch通路主要由受体(Notch1-4),配体(Jagged1-2,DLL1,3,4)和靶基因(Hairy族和Hes族)等共同构成。成熟的少突胶质细胞在中枢神经系统以及许多疾病中都扮演重要的角色。包绕中枢神经系统轴索形成髓鞘,并营养支持神经元,保持其功能的完整性。研究表明,在多发性硬化(multiple sclerosis,MS)的脱髓鞘区域,高表达于反应性星形胶质细胞的内皮素-1(endothelin-1,ET-1),可通过激活少突胶质细胞的Notch1信号通路,抑制少突胶质细胞分化及髓鞘化。ALS存在与MS相似的少突胶质细胞病理改变,包括少突胶质细胞变性、新生少突胶质细胞成熟障碍等。ALS小鼠脊髓中ET-1及Notch1信号通路是否也存在类似的改变呢?国内外鲜见报道。据此,该文将研究肌萎缩侧索硬化转基因小鼠不同时期腰髓少突胶质细胞改变及ET-1、Notch1胞内区(NICD)、Jagged-1、Delta-like 4(DLL4)以及Hes-1蛋白的表达变化及细胞定位情况,探讨ET-1及Notch1信号通路在ALS发病机制中的可能作用,为ALS治疗探寻新的可能靶点。方法:1转基因小鼠SOD1-G93A转基因小鼠在恒温、恒湿和无特定病原菌环境中饲养,给予灭菌的颗粒型鼠粮和无菌饮用水。动物实验过程按照河北省实验动物管理办法规定实行。以B6SJL-Tg(SOD1-93A)1Gur/J半合子雄鼠与B6SJLF1/J雌鼠交配繁殖、两种小鼠均购自美国Jackson实验室(Bar Harbor,ME,USA)。子代鼠在出生后一个月左右取尾尖部组织(2mm)提取DNA并经PCR扩增,鉴定是否携带有人突变SOD1基因。2转基因小鼠运动功能评分及分组2.1转基因小鼠运动功能评分从小鼠60天开始测量体重及进行转棒实验,每周2次,并进行运动功能评分。小鼠运动缺陷评分采用4分评价系统:1)正常,无运动障碍,无体重减轻:4分2)悬尾时后肢震颤:3分3)步态异常:2分4)至少一侧后肢瘫痪:1分5)小鼠仰或侧卧,30秒内不能翻正:0分2.2实验分组依据SOD1-93A小鼠的发病规律分为5组,分别为30天组,60天组,90天组,症状早期组及终末期组,对照组为90天非转基因小鼠。每组6只小鼠。症状早期指体重连续两次下降并出现步态异常,评分2分。终末期指侧卧30秒不能翻身,评分为0分。3实验方法3.1取材各时期小鼠在相应时间点取材。用10%的水合氯醛(350mg/Kg)腹腔内注射麻醉后,用PBS经左心室冲洗血液后,再用4%多聚甲醛灌流固定,取出脊髓组织置于固定液中4℃保存。3.2免疫组织化学法取一小段固定好的腰髓组织,浸泡在30%蔗糖中4℃过夜。腰膨大部分用O.C.T.Compound溶液包埋后,迅速放入液氮冷冻;使用LEICA CM1850冰冻切片机,切25μm厚切片;取完整腰髓切片放入0.01M PBS溶液;1%的H202(0.01MPBS溶液配)10分钟,封闭内源性过氧化物酶;0.01M PBS漂洗10分钟;5%的羊血清,0.3%Triton X-100(0.01M PBS溶液配)室温封闭打孔1小时;按浓度比在封闭液中分别加入一抗,4℃摇床过夜;PBST漂洗3次,每次10分钟后加入相应二抗,室温置于摇床1小时;PBST漂洗3次,每次10分钟;加入辣根过氧化物酶标记的链霉亲和素,室温置于摇床40分钟;PBST漂洗3次,每次10分钟;DAB显色,捞片,干燥后无水乙醇脱水,二甲苯透明,中性快干胶封片(湿封)。Olympus显微镜观察及照相。3.3免疫荧光法与免疫组织化学法类似,小鼠经过灌注、固定后取腰膨大处经LEICA CM1850冰冻切片,厚度为25μm。选取完整的腰髓切片,PBS漂洗2次,每次5分钟;1%Triton X-100(0.01M PBS溶液配)室温摇床30分钟;漂洗2分钟;5%羊血清、0.3%Triton X-100、2%奶粉(0.01M PBS配)封闭,加入相应的双标一抗,4°摇床过夜;0.01MPBS漂洗3次,每次10分钟;荧光二抗室温摇床避光孵育2小时;0.01MPBS漂洗3次,每次10分钟;捞片,含DAPI的抗荧光衰减封片剂封片;Olympus FV1000激光共聚焦显微镜观察及照相。结果:1 ALS小鼠腰髓中存在少突胶质细胞变性及少突胶质前体细胞增多。随病程进展,腰髓前角APC阳性少突胶质细胞进行性减少,NG2阳性少突胶质前体细胞进行性增多,以终末期为著。2 ALS小鼠腰髓ET-1阳性细胞随病程进展增多。ET-1主要表达于激活的星形胶质细胞。3随病程进展,ALS小鼠腰髓NICD阳性神经元减少,而NICD阳性少突胶质前体细胞增多。细胞核内可见NICD表达,以终末期最为明显。4随病程进展,ALS小鼠腰髓Jagged-1阳性细胞进行性增多,Jagged-1表达于小胶质细胞。5随病程进展,ALS小鼠腰髓DLL4阳性神经元减少,而DLL4阳性星形胶质细胞进行性增多。6 ALS小鼠腰髓Hes-1表达主要见于细胞核,随病程进展Hes-1阳性细胞进行性增多。结论:1 SOD1-G93A转基因小鼠腰髓存在少突胶质细胞变性及少突胶质前体细胞增生。2 SOD1-G93A转基因小鼠腰髓激活的星形胶质细胞表达ET-1。3 SOD1-G93A转基因小鼠腰髓存在Notch1激活及其配体、靶基因的表达升高。
[Abstract]:Objective: amyotrophic lateral sclerosis (amyotrophic lateral, sclerosis, ALS) is a involving cortex, brainstem, corticospinal tract and spinal motor neurons in neurodegenerative diseases, the main clinical manifestations were progressive muscle atrophy, leading to severe motor disability and respiratory muscle paralysis, the survival period is generally 3-5 years. According to the clinical statistics the incidence rate of 1.5-2.5/10 million.5%-10% for familial ALS, the remaining more than 90% belong to the pathogenesis of sporadic ALS. involved in inflammation, oxidative stress, protein aggregation, mitochondrial dysfunction, impaired axonal transport, glutamate excitotoxicity and neurotrophic factors such as lack of differentiation, migration pathway of.Notch pathogenesis of any single hypothesis not a good explanation of ALS in cell growth, outcome, and synaptic shaping and neuronal survival plays an important role in.Notch pathway mainly by receptor (Notc H1-4) (Jagged1-2, DLL1,3,4), the ligand and the target gene (Hairy group and Hes group) together. Mature oligodendrocytes have played an important role in the central nervous system and many diseases. Around the central nervous system axonal myelination, and nutritional support of neurons, keep the integrity of its function the study shows that in multiple sclerosis (multiple sclerosis, MS) demyelinating areas, endothelial overexpressed in reactive astrocytes in -1 (endothelin-1, ET-1), through the activation of the Notch1 signaling pathway of oligodendrocytes, myelin oligodendrocyte differentiation and inhibition of glial cells of.ALS are less pathological changes oligodendrocytes and MS similar, including oligodendrocyte degeneration, neonatal oligodendrocyte maturation disorder.ALS mice ET-1 in spinal Notch1 signaling pathways and whether there is a similar change? At home and abroad are rarely reported. Therefore, the This paper will study the transgenic mouse model of amyotrophic lateral sclerosis in different periods of spinal cord oligodendrocyte changes and ET-1, Notch1 cytoplasmic domain (NICD), Jagged-1, Delta-like 4 (DLL4) and Hes-1 protein expression changes and cellular localization, to investigate the possible role of ET-1 and Notch1 signaling pathway in the pathogenesis of ALS, explore may be a new target for the treatment of ALS. Methods: 1 transgenic mice of SOD1-G93A transgenic mice at constant temperature, humidity and feeding no specific pathogens in the environment, given the sterilization of the particle type rat food and sterile drinking water. Animal experiment process in accordance with the provisions of the measures for the administration of laboratory animal Hebei. With B6SJL-Tg (SOD1-93A) 1Gur/J hemizygote male B6SJLF1/J rats and female rats mated, two mice were purchased from American Jackson Laboratory (Bar Harbor, ME, USA). The offspring were born in about a month after the tail tip tissue (2mm) DNA was extracted and amplified by PCR, Kam Whether carrying human SOD1 gene mutation in.2 transgenic mouse movement function score and motor function score group 2.1 transgenic mice from mice 60 day body weight measurement and rotarod test, 2 times a week, and motor scores. The mice were scored with 4 points defects evaluation system: 1) normal, no movement disorders, no weight loss: 4 points 2) tail suspension when the hind limb tremor: 3 points 3) abnormal gait: 2 points 4) at least one hind limb paralysis: 1 points 5) mice back or side, 30 seconds is not over: 0 divided into 2.2 experimental groups according to the occurrence regularity of SOD1-93A mice were divided into 5 groups, respectively for 30 days group, 60 days group, 90 days group, onsetstage group and end-stage group, the control group was 90 days in non transgenic mice. 6 mice in each group. The early symptoms of weight two consecutive decline and the emergence of abnormal gait, score of 2. 30 seconds to end side can not stand up, 0 score,.3 experiment methods: 3.1. Each period of mice at the corresponding time point respectively. With 10% chloral hydrate (350mg/Kg) intraperitoneal injection of anesthesia, PBS after left ventricular blood after washing, then 4% paraformaldehyde perfusion fixation, spinal cord tissues placed in fixative preserved 4 C.3.2 immunohistochemical method for spinal cord segments fixed, soaked in 30% sucrose at 4 overnight. Most of lumbar enlargement with O.C.T.Compound solution after embedding, quickly placed in liquid nitrogen freezing; using LEICA CM1850 refrigerated microtome, cutting 25 m thick slices; take complete spinal cord slices in 0.01M PBS solution; 1% H202 (0.01MPBS solution) 10 minutes, closed egpo; 0.01M PBS rinse for 10 minutes; 5% sheep serum, 0.3%Triton X-100 (0.01M PBS solution) at room temperature for 1 hours by closed drilling; concentration ratio in blocking solution were added to the anti shaking 4 C overnight; 3 PBST rinse, every 10 minutes after adding phase Two resistance at room temperature in Table 1 hours; 3 PBST rinse, every 10 minutes; adding horseradish peroxidase labeled streptavidin at room temperature in table 40 minutes; 3 PBST rinse, every 10 minutes; DAB color, fishing, after drying, ethanol dehydration, xylene transparent, neutral adhesive mount (wet seal).Olympus microscope and camera.3.3 immunofluorescence and immunohistochemistry method similar to mice after perfusion, removed after fixation of lumbar enlargement by LEICA CM1850 frozen section, thickness of 25 M. lumbar spinal cord slice select complete PBS, rinse 2 times, each time for 5 minutes; 1%Triton X-100 (0.01M PBS solution with 30 minutes shaking at room temperature); rinse for 2 minutes; 5% sheep serum, 0.3%Triton X-100,2% (0.01M PBS) milk closed, add the corresponding double antibody, 4 ~ 3 0.01MPBS table for the night; rinse every 10 minutes; two fluorescent anti shaker dark incubation at room temperature for 2 hours; 0.01MPBS bleaching Wash 3 times, each time for 10 minutes; fishing, mounting medium containing DAPI by anti fluorescence decay; Olympus FV1000 laser confocal microscope and photographed. Results: oligodendrocyte degeneration and oligodendrocyte precursor cells increased 1 ALS in spinal cord of mice. With the progression of lumbar cord anterior horn of APC positive oligodendrocytes were decreased, NG2 positive oligodendrocyte precursor cells were increased in end-stage for.2 ALS in spinal cord of mice ET-1 positive cells increased with the progression of.ET-1 is mainly expressed in the astrocytes in the activation of.3 with the disease, reduce ALS in spinal cord of mice NICD positive neurons and NICD positive and less oligodendrocyte precursor cells increased. NICD was observed in the nucleus, with the most obvious end-stage.4 with the progression of ALS in spinal cord of mice Jagged-1 positive cells were increased, progress in microglia.5 with the course of the expression of Jagged-1 and ALS in spinal cord of mice DLL By 4 positive neurons, DLL4 positive astrocytes were increased.6 ALS in spinal cord of mice Hes-1 expression mainly in the nucleus, were increased with the progression of Hes-1 positive cells. Conclusion: the expression of ET-1.3 in lumbar spinal cord of SOD1-G93A transgenic mice Notch1 ligand activation and oligodendrocyte degeneration and oligodendrocyte precursor cell proliferation.2 SOD1-G93A transgenic mouse spinal cord astrocytes are 1 lumbar spinal cord of SOD1-G93A transgenic mice, increased the expression of target gene.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8

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