本文選題：syncytin-1　切入點(diǎn)：運(yùn)動(dòng)神經(jīng)元　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:運(yùn)動(dòng)神經(jīng)元病(motor neuron disease,MND)是一種致命性神經(jīng)系統(tǒng)變性疾病,迄今病因不明。人內(nèi)源性逆轉(zhuǎn)錄病毒-W家族e(cuò)nv基因編碼的糖蛋白syncytin-1在運(yùn)動(dòng)神經(jīng)元病患者活檢骨骼肌組織中異常高表達(dá)。實(shí)驗(yàn)研究發(fā)現(xiàn)小鼠后肢骨骼肌組織內(nèi)syncytin-1的高表達(dá)可誘導(dǎo)脊髓腰膨大部位前角運(yùn)動(dòng)神經(jīng)元損傷,但機(jī)制尚不清楚。目的:研究谷氨酸興奮性毒性在小鼠后肢骨骼肌高表達(dá)syncytin-1誘導(dǎo)脊髓和運(yùn)動(dòng)皮質(zhì)運(yùn)動(dòng)神經(jīng)元損傷中的作用。方法:選取8周大的C57BL/6J雄鼠隨機(jī)分為實(shí)驗(yàn)組1、實(shí)驗(yàn)組2和對(duì)照組,實(shí)驗(yàn)組1、實(shí)驗(yàn)組2左側(cè)脛前肌分別注射100μl、200μl重組質(zhì)粒p CMV-tag2B-syncytin(0.8μg/μl),對(duì)照組注射200μl空載質(zhì)粒p CMV-tag2B(0.8μg/μl)。予以10%水合氯醛腹腔注射麻醉,暴露左側(cè)脛前肌,注射質(zhì)粒后每月測(cè)量并記錄小鼠體重,在注射質(zhì)粒4個(gè)月后評(píng)估小鼠運(yùn)動(dòng)功能;進(jìn)行神經(jīng)肌肉電生理檢測(cè);HE染色觀察腓腸肌肌纖維的形態(tài);尼氏染色觀察運(yùn)動(dòng)皮層和脊髓腰膨大部位前角運(yùn)動(dòng)神經(jīng)元數(shù)目和形態(tài);通過(guò)免疫熒光染色的方法,分別觀察小鼠運(yùn)動(dòng)皮層和脊髓腰膨大部位星形膠質(zhì)細(xì)胞骨架蛋白-膠質(zhì)纖維酸性蛋白(Glial Fibrillary Acidic Protein,GFAP);通過(guò)Western blot方法、RT-PCR分別在蛋白水平及基因水平檢測(cè)小鼠運(yùn)動(dòng)皮層及脊髓腰膨大部位syncytin-1、α-氨基-3-羧基-5-甲基異惡唑-4-丙酸(α-amino-3-hydroxyl-5-methyl-4-isox-azolepropionic acid receptors,AMPA)受體Glu R2亞型、谷氨酸轉(zhuǎn)運(yùn)體1(Glutamate transporter 1,GLT-1)的表達(dá)量。結(jié)果:注射質(zhì)粒4個(gè)月后,3組小鼠體重?zé)o明顯差別(F=0.51,P㧐0.05);爬鐵絲網(wǎng)實(shí)驗(yàn)顯示,實(shí)驗(yàn)組1、2均較對(duì)照組小鼠爬鐵絲網(wǎng)時(shí)間延長(zhǎng),但差別無(wú)統(tǒng)計(jì)學(xué)意義(F=3.37,P㧐0.05)。神經(jīng)肌肉電生理檢測(cè)顯示:實(shí)驗(yàn)組1、2小鼠腸肌靜息時(shí)出現(xiàn)纖顫電位,輕收縮時(shí)動(dòng)作電位波幅增高、時(shí)限延長(zhǎng),大力收縮時(shí)復(fù)合肌肉動(dòng)作電位呈現(xiàn)典型的失神經(jīng)支配的單純相;HE染色顯示實(shí)驗(yàn)組1、2小鼠腓腸肌橫斷面部分肌纖維萎縮,肌群周徑較對(duì)照組減小,部分肌纖維呈多角形萎縮等典型神經(jīng)源性損害的表現(xiàn);尼氏染色顯示:實(shí)驗(yàn)組1、2小鼠腰髓前角運(yùn)動(dòng)神經(jīng)元數(shù)目顯著減少(F=23.37,P0.05),實(shí)驗(yàn)組2比實(shí)驗(yàn)組1運(yùn)動(dòng)神經(jīng)元減少更為明顯(q=3.27,P=0.01)。實(shí)驗(yàn)組1、2小鼠運(yùn)動(dòng)皮層運(yùn)動(dòng)神經(jīng)元數(shù)量顯著減少(F=224.96;P0.001),實(shí)驗(yàn)組2較實(shí)驗(yàn)組1減少更為明顯(q=12.76,P㩳0.001)。免疫熒光染色顯示:實(shí)驗(yàn)組1、實(shí)驗(yàn)組2運(yùn)動(dòng)皮層和脊髓腰膨大部位GFAP表達(dá)量增多且呈激活狀態(tài);PCR及western blot示:GLT-1、AMPA(Glu R2)在運(yùn)動(dòng)皮層和脊髓腰膨大部位的相對(duì)表達(dá)量在實(shí)驗(yàn)組1、2均較對(duì)照組顯著減少(均P㩳0.05);Syncytin-1蛋白及m RNA相對(duì)表達(dá)量在實(shí)驗(yàn)組1、2均較對(duì)照組顯著升高(均P㩳0.05)。結(jié)論:1.小鼠后肢骨骼肌syncytin-1高表達(dá)可以誘導(dǎo)小鼠運(yùn)動(dòng)皮層和脊髓前角運(yùn)動(dòng)神經(jīng)元損傷,且損傷程度與骨骼肌syncytin-1表達(dá)水平呈正相關(guān)。2.谷氨酸興奮性毒性作用及星形膠質(zhì)細(xì)胞激活可能參與syncytin-1誘導(dǎo)運(yùn)動(dòng)神經(jīng)元損傷病理過(guò)程,其中谷氨酸興奮性毒性的產(chǎn)生可能通過(guò)減少谷氨酸轉(zhuǎn)運(yùn)體GLT-1使突觸間隙谷氨酸積聚和減少突觸后膜AMPA受體Glu R2亞型增加鈣離子通透性兩條途徑造成運(yùn)動(dòng)神經(jīng)元損傷。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R744.8

【參考文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王若紅;Syncytin在小鼠失神經(jīng)支配腓腸肌中的表達(dá)[D];天津醫(yī)科大學(xué);2012年

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