家族型肌萎缩侧索硬化动物模型脊髓侧角神经元的特征

发布时间：2018-06-13 22:21

本文选题：脊髓侧角 + 乙酰胆碱酯酶　；参考：《河北医科大学》2012年硕士论文

【摘要】：目的：肌萎缩侧索硬化（amyotrophic lateral sclerosis，ALS）是一种病因未明神经系统变性病，选择性损伤大脑皮层运动神经元、脑干运动神经核神经元、脊髓前角a运动神经元，导致进行性加重的肌肉无力、萎缩，最终因呼吸肌麻痹死亡。近年来研究发现部分ALS患者存在自主神经系统、锥体外系系统、感觉系统等非运动神经区域受累的症状，提示ALS可能是一个多系统累及的神经变性疾病。Murata Y发现ALS伴发昼夜血压的变化,血压与心率之间的相关性的降低，严重可诱发循环衰竭、心律失常乃至猝死[1]；及其他自主神经异常的表现如：排汗增加及减少[2]、唾液分泌减少[3]、胃排空时间延长[4]等改变。 Takahashi H及Kennedy PG分别报道ALS患者胸髓中间外侧核神经元的丢失[5-6],Itoh T发现神经元免疫活性的改变[7]及Kihira T也在萎缩神经元胞浆内发现了包涵体[8]，比如Onuf,s核的bunnia小体。在SOD1(V118L)基因突变的家族型ALS患者短期内出现自主神经衰竭，尸检发现延髓自主神经核及中间外侧柱明显的神经元丢失[9]。对SOD1~(G93A)转基因小鼠研究发现安静状态下早期转基因鼠心率增高，提示疾病早期自主神经功能损伤[10]。交感神经的低级中枢位于脊髓胸1（T1）至腰2-腰3（L2或L3），组成了中间外侧柱(IML)，在调节自主神经功能方面具有重要作用。本实验拟应用甲苯胺蓝染色及免疫组化的方法，观察家族型肌萎缩侧索硬化动物模型（SOD1~(G93A)转基因小鼠）病程不同阶段脊髓侧角神经元的结构及乙酰胆碱酯酶的表达情况，旨在探讨脊髓侧角神经元在ALS的发病过程的变化特征，为ALS发病机制研究提供一定的理论基础。方法： 1模型建立及分组饲养繁殖家族型肌萎缩侧索硬化动物模型SOD1~(G93A)转基因小鼠，参考Vercelli A[11]等的1-5分评分法进行评分,4分为发病时间，1分为死亡时间,60天为症状前期，4分为症状早期，1分为终末期。各对照组为同窝、同月龄未转人突变的SOD1基因的小鼠。每组各3只小鼠。 2取材 10%水合氯醛350mg/kg腹腔注射麻醉后，心脏灌注4%多聚甲醛固定20min，取小鼠脊髓胸段将其分成上段（T1-T3）、中段（T4-T6）、下段（T7-T9），分别以4%多聚甲醛固定48h。 3甲苯胺蓝染色后固定的组织块经梯度酒精脱水，二甲苯透明，石蜡包埋，连续组织切5μm片，进行甲苯胺蓝染色。依据Oppenheimer定量组织学方法[12]，计数脊髓侧角神经元。 4免疫组化染色 10%水合氯醛350mg/kg腹腔注射麻醉后，心脏灌注4%多聚甲醛固定30min，取小鼠脊髓胸段将其分成上胸髓（T1-T3）、中胸髓（T4-T6）、下胸髓（T7-T9），分别以4%多聚甲醛固定12小时，，震荡切片25μm厚，应用乙酰胆碱酯酶进行免疫组化染色。 5统计学处理采用SPSS13.0统计学软件对数据进行统计处理。采用均数±标准差表示上段（T1-T3）、中段（T4-T6）、下段（T7-T9）各脊髓切片两侧神经元计数之和，三组数据的比较采用单因素方差分析，两组数据的比较采用两个独立样本比较的t检验，以a=0.05为显著性检验标准。结果： 1模型建立 B6SJL-BTg（SOD1~(G93A)）1Gur/J半合子雄鼠与B6SJLF1/J+/+雌鼠1:1杂交，在恒温、恒湿、12h光照/黑暗交替循环、无特殊病原菌的（Specificpathogen free， SPF）环境中，喂以灭菌的SPF级颗粒型鼠类饲料及灭菌水，经剪尾尖提取DNA、PCR扩增后、琼脂糖凝胶电泳结果示（Fig.1）：位于200-300bp之间的条带（236bp）为mSOD1的PCR产物，此种小鼠为SOD1~(G93A)转基因阳性小鼠。没有此条带为非mSOD1的PCR产物，为非SOD1~(G93A)转基因鼠。 2甲苯胺蓝染色 2.1上胸髓（T1-T3）症状前期、发病期、终末期SOD1~(G93A)转基因小鼠中神经元计数分别为7.4±3.0、7.0±2.0、3.7±1.5，对照组分别为5.3±1.9、4.1±1.2、6.1±1.9，症状前期和发病期期较终末期比较均有显著性差别（P0.05）。发病期及终末期神经元计数较对照组有显著性差别（P0.05）。症状前期及发病期转基因鼠神经元大致正常，终末期神经元变性。 2.2中胸髓（T4-T6） SOD1~(G93A)转基因小鼠症状前期、发病期、终末期神经元计数分别为4.5±1.6、4.3±1.5、2.5±1.2，总体均数比较P=0.01(0.05）差别无显著性意义。对照组分别为4.1±2.0、4.5±2.0、5.5±1.6，终末期SOD1~(G93A)转基因小鼠神经元细胞计数较对照组有显著性差别（P0.05）。症状前期及发病期转基因鼠神经元大致正常，终末期神经元变性。 2.3下胸髓（T7-T9） SOD1~(G93A)转基因小鼠在症状前期、发病期、终末期神经元计数分别为4.4±1.6、5.0±1.8、2.4±0.8，对照组分别为5.0±2.0、4.4±1.2、5.5±1.6，症状前期和发病期较终末期比较均有显著性差别（P0.05）。终末期SOD1~(G93A)转基因小鼠神经元细胞计数较对照组有显著性差别（P0.05）。症状前期及发病期转基因鼠神经元大致正常，终末期神经元变性。 3免疫组化染色 3.1上胸髓（T1-T3） SOD1~(G93A)转基因小鼠症状前期、发病期、终末期神经元计数分别为6.5±1.2、6.0±2.0、3.7±1.2，对照组分别为5.8±1.9、7.8±1.5、5.0±1.0，症状前期和发病期较终末期比较均有差别（P0.05）。发病期神经元细胞计数较对照组有显著性差别（P0.05）。症状前期及发病期神经元包浆呈深褐色，胞核无色，成多边形。终末期神经元变性，着色变淡。 3.2中胸髓（T4-T6） SOD1~(G93A)转基因小鼠症状前期、发病期、终末期神经元计数分别为6.6±2.6、4.9±2.0、3.0±1.4，症状前期和发病期与终末比较均有差别（P0.05）。对照组分别为4.8±1.0、5.5±0.6、5.8±1.0，终末期神经元细胞计数较对照组有显著性差别（P0.05）。症状前期及发病期神经元包浆呈深褐色，胞核无色，成多边形。终末期神经元变性，着色变淡。 3.3下胸髓（T7-T9） SOD1~(G93A)转基因小鼠症状前期、发病期、终末期神经元计数分别为6.7±1.8、6.3±2.6、3.0±1.0，总体均数比较P=0.059(0.05）差别无显著性意义,对照组分别为5.3±1.2、5.3±1.5、5.0±0.0，终末期神经元细胞计数较对照组有显著性差别（P0.05）。症状前期及发病期神经元包浆呈深褐色，胞核无色，成多边形。终末期神经元变性，着色变淡。结论：在SOD1转基因鼠发病过程中，存在脊髓侧角神经元丢失，尤其是在疾病的终末期，上、中、下胸髓节段间无明显差异。
[Abstract]:Objective : Amyotrophic lateral sclerosis ( ALS ) is a kind of motor neuron , brain stem motor neuron , spinal cord anterior horn a motor neuron , which leads to progressive increase of muscle weakness , atrophy and eventual death due to respiratory muscle paralysis .
The abnormal manifestations of other autonomic nervous abnormalities were as follows : increased perspiration and decreased edema , decreased saliva secretion , decreased gastric emptying time , and prolonged gastric emptying time , and so on .

In the case of familial ALS patients with mutation of SOD1 ( V118L ) gene mutation , spontaneous nerve failure occurred in the short term of familial ALS patients with gene mutation of SOD1 ( V118L ) . In the study of transgenic mice SOD1 ~ ( G93A ) , it was found that the heart rate of transgenic mice was increased in the early stage of the disease , which suggested that the early autonomic nervous function could be damaged in the early stage of the disease .

The lower center of sympathetic nerve is located in the spinal cord chest 1 ( T1 ) to the waist 2 - waist 3 ( L2 or L3 ) , which forms the medial outer column ( IML ) , which plays an important role in regulating the function of the autonomic nervous system .

In this experiment , we studied the structure of spinal dorsal horn neurons and the expression of acetylcholine esterase in different stages of the disease course of familial myotrophic lateral sclerosis animal model ( SOD1 ~ ( G93A ) transgenic mice ) by using the method of methylene blue staining and immunohistochemistry .

Method :

1 Model establishment and grouping

The model SOD1 ~ ( G93A ) transgenic mice were scored by means of 1 - 5 scoring method , such as Vercelli A , 11 , and so on . The rats were divided into four groups : the time of onset , the time of death , the early stage of 60 days , and the early stage of the symptom .

2 Materials

After intraperitoneal injection of 350mg / kg water chloral hydrate ( 350mg / kg ) , the hearts were perfused with 4 % polyformaldehyde for 20min , and the thoracic spinal segments of the spinal cord were divided into upper segment ( T1 - T3 ) , middle segment ( T4 - T6 ) and lower segment ( T7 - 9 ) , respectively , and fixed for 48 hours with 4 % polyformaldehyde .

3 - Methylphenamine blue staining

The posterior fixed tissue mass was dehydrated by gradient alcohol , transparent in xylene , paraffin - embedded , and cut into 5 渭m continuous tissue .

4 Immunohistochemical staining

After intraperitoneal injection of 350mg / kg water chloral hydrate ( 350mg / kg ) , the hearts were perfused with 4 % polyformaldehyde for 30 min . The thoracic spinal segments of the spinal cord were divided into upper thoracic spinal cord ( T1 - T3 ) , middle thoracic spinal cord ( T4 - T6 ) and lower thoracic spinal cord ( T7 - 9 ) respectively .

5 Statistical Processing

The data were statistically analyzed by SPSS 13.0 . The mean 卤 standard deviation was used to represent the sum of neuron counts on both sides of the upper segment ( T1 - T3 ) , middle section ( T4 - T6 ) and lower segment ( T7 - 9 ) . The comparison of three groups of data was analyzed by single factor analysis . The comparison between two groups of data was t - test compared with two independent samples , with a = 0.05 as the significance test standard .

Results :

1 Model Establishment

The results showed that the PCR products of non - SOD1 - ( G93A ) transgenic mice were non - SOD1 - ( G93A ) transgenic mice .

2 - Methylphenamine blue staining

2.1 Upper Thoracic Pulp ( T1 - T3 )

The number of neurons in transgenic mice were 7.4 卤 3.0 , 7.0 卤 2.0 , 3.7 卤 1.5 , 5.3 卤 1.9 , 4.1 卤 1.2 , 6.1 卤 1.9 in the control group , respectively .

2.2 Middle thoracic spinal cord ( T4 - T6 )

There was no significant difference between SOD1 ~ ( G93A ) transgenic mice at the early stage , the onset stage and the end - stage neuron count ( 4.5 卤 1.6 , 4.3 卤 1.5 , 2.5 卤 1.2 , P = 0.01 ( 0.05 )) . The control group was 4.1 卤 2.0 , 4.5 卤 2.0 , 5.5 卤 1.6 , and the end - stage SOD1 ~ ( G93A ) transgenic mice were significantly different from the control group ( P0.05 ) .

2.3 Lower thoracic spinal cord ( T7 - T8 )

SOD1 ~ ( G93A ) transgenic mice were 4.4 卤 1.6 , 5.0 卤 1.8 , 2.4 卤 0.8 , 5.0 卤 2.0 , 4.4 卤 1.2 , 5.5 卤 1.6 respectively . There was significant difference between the end - stage SOD1 ~ ( G93A ) transgenic mice .

3 Immunohistochemical staining

3.1 Upper Thoracic Pulp ( T1 - T3 )

There was a significant difference in the number of neuron cells in the early stage and the onset stage ( P0.05 ) . The neuronal cell counts were dark brown in the early stage of the symptoms and the nuclei were colorless and polygonal . The end - stage neurons were denatured and the coloration was pale .

3.2 Middle thoracic spinal cord ( T4 - T6 )

In the early stage of the symptoms , the count of the end - stage neurons was 6.6 卤 2.6 , 4.9 卤 2.0 , 3.0 卤 1.4 , and there was a significant difference between the early stage and the end - stage neuron count ( P < 0.05 ) . The control group was 4.8 卤 1.0 , 5.5 卤 0.6 , 5.8 卤 1.0 , and the end - stage neuronal cell count was significantly different from that of the control group ( P0.05 ) .

3.3 Lower thoracic spinal cord ( T7 - T8 )

There was no significant difference between SOD1 ~ ( G93A ) transgenic mice at the early stage , the onset stage and the end - stage neuron count of 6.7 卤 1.8 , 6.3 卤 2.6 , 3.0 卤 1.0 , P = 0.059 ( 0.05 ) , respectively . The control group was 5.3 卤 1.2 , 5.3 卤 1.5 , 5.0 卤 0.0 , and the end - stage neuronal cell count was significantly different from that of the control group ( P0.05 ) .

Conclusion : There is no significant difference in the dorsal horn neurons of the spinal cord during the pathogenesis of SOD1 transgenic mice , especially at the end of the disease , in the upper , middle and lower thoracic spinal segments .
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R744;R-332

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