谷氨酸兴奋性毒性在syncytin-1诱导运动神经元损伤中的作用

发布时间：2018-03-21 00:20

本文选题：syncytin-1　切入点：运动神经元　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:运动神经元病(motor neuron disease,MND)是一种致命性神经系统变性疾病,迄今病因不明。人内源性逆转录病毒-W家族env基因编码的糖蛋白syncytin-1在运动神经元病患者活检骨骼肌组织中异常高表达。实验研究发现小鼠后肢骨骼肌组织内syncytin-1的高表达可诱导脊髓腰膨大部位前角运动神经元损伤,但机制尚不清楚。目的:研究谷氨酸兴奋性毒性在小鼠后肢骨骼肌高表达syncytin-1诱导脊髓和运动皮质运动神经元损伤中的作用。方法:选取8周大的C57BL/6J雄鼠随机分为实验组1、实验组2和对照组,实验组1、实验组2左侧胫前肌分别注射100μl、200μl重组质粒p CMV-tag2B-syncytin(0.8μg/μl),对照组注射200μl空载质粒p CMV-tag2B(0.8μg/μl)。予以10%水合氯醛腹腔注射麻醉,暴露左侧胫前肌,注射质粒后每月测量并记录小鼠体重,在注射质粒4个月后评估小鼠运动功能;进行神经肌肉电生理检测;HE染色观察腓肠肌肌纤维的形态;尼氏染色观察运动皮层和脊髓腰膨大部位前角运动神经元数目和形态;通过免疫荧光染色的方法,分别观察小鼠运动皮层和脊髓腰膨大部位星形胶质细胞骨架蛋白-胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP);通过Western blot方法、RT-PCR分别在蛋白水平及基因水平检测小鼠运动皮层及脊髓腰膨大部位syncytin-1、α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(α-amino-3-hydroxyl-5-methyl-4-isox-azolepropionic acid receptors,AMPA)受体Glu R2亚型、谷氨酸转运体1(Glutamate transporter 1,GLT-1)的表达量。结果:注射质粒4个月后,3组小鼠体重无明显差别(F=0.51,P㧐0.05);爬铁丝网实验显示,实验组1、2均较对照组小鼠爬铁丝网时间延长,但差别无统计学意义(F=3.37,P㧐0.05)。神经肌肉电生理检测显示:实验组1、2小鼠肠肌静息时出现纤颤电位,轻收缩时动作电位波幅增高、时限延长,大力收缩时复合肌肉动作电位呈现典型的失神经支配的单纯相;HE染色显示实验组1、2小鼠腓肠肌横断面部分肌纤维萎缩,肌群周径较对照组减小,部分肌纤维呈多角形萎缩等典型神经源性损害的表现;尼氏染色显示:实验组1、2小鼠腰髓前角运动神经元数目显著减少(F=23.37,P0.05),实验组2比实验组1运动神经元减少更为明显(q=3.27,P=0.01)。实验组1、2小鼠运动皮层运动神经元数量显著减少(F=224.96;P0.001),实验组2较实验组1减少更为明显(q=12.76,P㩳0.001)。免疫荧光染色显示:实验组1、实验组2运动皮层和脊髓腰膨大部位GFAP表达量增多且呈激活状态;PCR及western blot示:GLT-1、AMPA(Glu R2)在运动皮层和脊髓腰膨大部位的相对表达量在实验组1、2均较对照组显著减少(均P㩳0.05);Syncytin-1蛋白及m RNA相对表达量在实验组1、2均较对照组显著升高(均P㩳0.05)。结论:1.小鼠后肢骨骼肌syncytin-1高表达可以诱导小鼠运动皮层和脊髓前角运动神经元损伤,且损伤程度与骨骼肌syncytin-1表达水平呈正相关。2.谷氨酸兴奋性毒性作用及星形胶质细胞激活可能参与syncytin-1诱导运动神经元损伤病理过程,其中谷氨酸兴奋性毒性的产生可能通过减少谷氨酸转运体GLT-1使突触间隙谷氨酸积聚和减少突触后膜AMPA受体Glu R2亚型增加钙离子通透性两条途径造成运动神经元损伤。
[Abstract]:Background: motor neuron disease (motor neuron, disease, MND) is a fatal neurodegenerative disease of unknown etiology. By encoding human endogenous retrovirus -W env gene family of glycoprotein syncytin-1 in patients with motor neuron disease of skeletal muscle biopsy tissue abnormal high expression. The experimental study found that high expression of syncytin-1 in mouse hindlimb skeletal muscle tissue the motor neurons in anterior horn of spinal cord induced by injury, but the mechanism is not clear. Objective: To study the expression of glutamate excitotoxicity in mouse hindlimb skeletal muscle induced by syncytin-1 spinal cord and motor cortex of motor neuron injury. Methods: 8 week old male C57BL/6J rats were randomly divided into experimental group 1, experimental group 2 and control group, experimental group 1, experimental group 2 left anterior tibial muscle were injected with 100 L and 200 L recombinant plasmid P CMV-tag2B-syncytin (0.8 g/ L), the control group was injected with 20 0 L P CMV-tag2B plasmid (0.8 g/ L). By intraperitoneal injection of 10% chloral hydrate anesthesia, to expose the left anterior tibial muscle, after injection of plasmid monthly measured and recorded body weight in mice, in 4 months after the injection of plasmid were evaluated on nerve muscle motor function; electrophysiology; HE staining observation of gastrocnemius muscle fiber morphology; observe the number and morphology of motor neurons of anterior horn motor cortex and spinal cord Nissl staining method; by immunofluorescence staining, were observed in mice of motor cortex and spinal cord astrocyte cytoskeletal protein - glial fibrillary acidic protein (Glial Fibrillary Acidic Protein, GFAP) by Western blot; methods RT-PCR, syncytin-1 respectively at the protein level and gene level detection of mouse motor cortex and spinal cord, alpha amino -3- carboxy methyl isoxazole propionate (-5- -4- alpha -amino-3-hydroxyl-5-methy L-4-isox-azolepropionic acid receptors, AMPA Glu R2) receptor subtype of glutamate transporter 1 (Glutamate transporter 1, GLT-1). Results: the expression of plasmid injection after 4 months, there was no significant difference in body weight of 3 groups of mice (F=0.51, P? 0.05); climbing the barbed wire display experiment, the experimental group of 1,2 mice than in the control group climb the fence is prolonged, but the difference was not statistically significant (F=3.37, P? 0.05). Neuromuscular electrophysiology: fibrillation potential experimental group of 1,2 mice intestinal resting muscle, increased the amplitude of action potential in light contraction duration, when strong contraction of compound muscle action potentials showed typical denervation simple phase; HE staining showed that the experimental group of 1,2 mice gastrocnemius muscle cross-sectional part of muscle fiber atrophy, muscle circumference decreased compared with the control group, part of the muscle fiber atrophy were polygonal typical neurogenic damage performance; Nissl staining showed that: the experimental group 1 2, the number of motor neurons in anterior horn of spinal cord of mice decreased significantly (F=23.37, P0.05), 2 in the experimental group than the experimental group 1 motor neurons decreased more significantly (q=3.27, P=0.01). The experimental group of 1,2 mice significantly reduced the number of motor neurons in the motor cortex (F=224.96; P0.001), 2 in the experimental group than in the experimental group reduced more 1 obviously (q=12.76, P? 0.001). Immunofluorescence staining showed that the experimental group 1, experimental group 2 motor cortex and spinal cord GFAP expression increased and the activation of PCR and Western blot; AMPA: GLT-1 (Glu, R2) in the relative expression of motor cortex and spinal cord in the lumbar enlargement of parts 1,2 in the experimental group were significantly lower than those in control group (P? 0.05); Syncytin-1 protein and m RNA expression in 1,2 of experimental group was significantly higher than the control group (P? 0.05). Conclusion: high expression of 1. mouse hindlimb skeletal muscle syncytin-1 could induce motor cortex and spinal cord of God After injury, and the degree of damage and syncytin-1 expression in skeletal muscle activation level of.2. was positively related to glutamate excitotoxicity and astrocytes may be involved in syncytin-1 induced motor neuron injury pathological process, which may produce glutamate excitotoxicity by reducing glutamate transporter GLT-1 in the synaptic cleft glutamate accumulation and reduction of postsynaptic membrane receptor AMPA Glu R2 subtype increased calcium ion permeability of two pathways caused by motor neuron injury.

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

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