本文選題：Notch1信號通路　切入點(diǎn)：少突膠質(zhì)前體細(xì)胞　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:肌萎縮側(cè)索硬化癥(amyotrophic lateral sclerosis,ALS)是一種累及皮層、腦干、皮質(zhì)脊髓束以及脊髓運(yùn)動(dòng)神經(jīng)元的神經(jīng)變性疾病,主要的臨床表現(xiàn)為進(jìn)行性肌肉萎縮,導(dǎo)致運(yùn)動(dòng)功能嚴(yán)重障礙甚至呼吸肌麻痹,患者生存期一般為3-5年。據(jù)臨床統(tǒng)計(jì)其發(fā)病率為1.5-2.5/10萬。5%-10%為家族性ALS,其余90%以上屬于散發(fā)型ALS。其發(fā)病機(jī)理涉及炎癥、氧化應(yīng)激、蛋白聚集、線粒體功能障礙、軸漿運(yùn)輸受損、谷氨酸興奮性毒和神經(jīng)營養(yǎng)因子不足等,任何單一假說都無法很好的解釋ALS的發(fā)病機(jī)制。Notch信號通路在決定細(xì)胞結(jié)局、遷移分化、生長以及突觸的塑造和神經(jīng)元存活中扮演重要的角色。Notch通路主要由受體(Notch1-4),配體(Jagged1-2,DLL1,3,4)和靶基因(Hairy族和Hes族)等共同構(gòu)成。成熟的少突膠質(zhì)細(xì)胞在中樞神經(jīng)系統(tǒng)以及許多疾病中都扮演重要的角色。包繞中樞神經(jīng)系統(tǒng)軸索形成髓鞘,并營養(yǎng)支持神經(jīng)元,保持其功能的完整性。研究表明,在多發(fā)性硬化(multiple sclerosis,MS)的脫髓鞘區(qū)域,高表達(dá)于反應(yīng)性星形膠質(zhì)細(xì)胞的內(nèi)皮素-1(endothelin-1,ET-1),可通過激活少突膠質(zhì)細(xì)胞的Notch1信號通路,抑制少突膠質(zhì)細(xì)胞分化及髓鞘化。ALS存在與MS相似的少突膠質(zhì)細(xì)胞病理改變,包括少突膠質(zhì)細(xì)胞變性、新生少突膠質(zhì)細(xì)胞成熟障礙等。ALS小鼠脊髓中ET-1及Notch1信號通路是否也存在類似的改變呢?國內(nèi)外鮮見報(bào)道。據(jù)此,該文將研究肌萎縮側(cè)索硬化轉(zhuǎn)基因小鼠不同時(shí)期腰髓少突膠質(zhì)細(xì)胞改變及ET-1、Notch1胞內(nèi)區(qū)(NICD)、Jagged-1、Delta-like 4(DLL4)以及Hes-1蛋白的表達(dá)變化及細(xì)胞定位情況,探討ET-1及Notch1信號通路在ALS發(fā)病機(jī)制中的可能作用,為ALS治療探尋新的可能靶點(diǎn)。方法:1轉(zhuǎn)基因小鼠SOD1-G93A轉(zhuǎn)基因小鼠在恒溫、恒濕和無特定病原菌環(huán)境中飼養(yǎng),給予滅菌的顆粒型鼠糧和無菌飲用水。動(dòng)物實(shí)驗(yàn)過程按照河北省實(shí)驗(yàn)動(dòng)物管理辦法規(guī)定實(shí)行。以B6SJL-Tg(SOD1-93A)1Gur/J半合子雄鼠與B6SJLF1/J雌鼠交配繁殖、兩種小鼠均購自美國Jackson實(shí)驗(yàn)室(Bar Harbor,ME,USA)。子代鼠在出生后一個(gè)月左右取尾尖部組織(2mm)提取DNA并經(jīng)PCR擴(kuò)增,鑒定是否攜帶有人突變SOD1基因。2轉(zhuǎn)基因小鼠運(yùn)動(dòng)功能評分及分組2.1轉(zhuǎn)基因小鼠運(yùn)動(dòng)功能評分從小鼠60天開始測量體重及進(jìn)行轉(zhuǎn)棒實(shí)驗(yàn),每周2次,并進(jìn)行運(yùn)動(dòng)功能評分。小鼠運(yùn)動(dòng)缺陷評分采用4分評價(jià)系統(tǒng):1)正常,無運(yùn)動(dòng)障礙,無體重減輕:4分2)懸尾時(shí)后肢震顫:3分3)步態(tài)異常:2分4)至少一側(cè)后肢癱瘓:1分5)小鼠仰或側(cè)臥,30秒內(nèi)不能翻正:0分2.2實(shí)驗(yàn)分組依據(jù)SOD1-93A小鼠的發(fā)病規(guī)律分為5組,分別為30天組,60天組,90天組,癥狀早期組及終末期組,對照組為90天非轉(zhuǎn)基因小鼠。每組6只小鼠。癥狀早期指體重連續(xù)兩次下降并出現(xiàn)步態(tài)異常,評分2分。終末期指側(cè)臥30秒不能翻身,評分為0分。3實(shí)驗(yàn)方法3.1取材各時(shí)期小鼠在相應(yīng)時(shí)間點(diǎn)取材。用10%的水合氯醛(350mg/Kg)腹腔內(nèi)注射麻醉后,用PBS經(jīng)左心室沖洗血液后,再用4%多聚甲醛灌流固定,取出脊髓組織置于固定液中4℃保存。3.2免疫組織化學(xué)法取一小段固定好的腰髓組織,浸泡在30%蔗糖中4℃過夜。腰膨大部分用O.C.T.Compound溶液包埋后,迅速放入液氮冷凍;使用LEICA CM1850冰凍切片機(jī),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分鐘,封閉內(nèi)源性過氧化物酶;0.01M PBS漂洗10分鐘;5%的羊血清,0.3%Triton X-100(0.01M PBS溶液配)室溫封閉打孔1小時(shí);按濃度比在封閉液中分別加入一抗,4℃搖床過夜;PBST漂洗3次,每次10分鐘后加入相應(yīng)二抗,室溫置于搖床1小時(shí);PBST漂洗3次,每次10分鐘;加入辣根過氧化物酶標(biāo)記的鏈霉親和素,室溫置于搖床40分鐘;PBST漂洗3次,每次10分鐘;DAB顯色,撈片,干燥后無水乙醇脫水,二甲苯透明,中性快干膠封片(濕封)。Olympus顯微鏡觀察及照相。3.3免疫熒光法與免疫組織化學(xué)法類似,小鼠經(jīng)過灌注、固定后取腰膨大處經(jīng)LEICA CM1850冰凍切片,厚度為25μm。選取完整的腰髓切片,PBS漂洗2次,每次5分鐘;1%Triton X-100(0.01M PBS溶液配)室溫?fù)u床30分鐘;漂洗2分鐘;5%羊血清、0.3%Triton X-100、2%奶粉(0.01M PBS配)封閉,加入相應(yīng)的雙標(biāo)一抗,4°搖床過夜;0.01MPBS漂洗3次,每次10分鐘;熒光二抗室溫?fù)u床避光孵育2小時(shí);0.01MPBS漂洗3次,每次10分鐘;撈片,含DAPI的抗熒光衰減封片劑封片;Olympus FV1000激光共聚焦顯微鏡觀察及照相。結(jié)果:1 ALS小鼠腰髓中存在少突膠質(zhì)細(xì)胞變性及少突膠質(zhì)前體細(xì)胞增多。隨病程進(jìn)展,腰髓前角APC陽性少突膠質(zhì)細(xì)胞進(jìn)行性減少,NG2陽性少突膠質(zhì)前體細(xì)胞進(jìn)行性增多,以終末期為著。2 ALS小鼠腰髓ET-1陽性細(xì)胞隨病程進(jìn)展增多。ET-1主要表達(dá)于激活的星形膠質(zhì)細(xì)胞。3隨病程進(jìn)展,ALS小鼠腰髓NICD陽性神經(jīng)元減少,而NICD陽性少突膠質(zhì)前體細(xì)胞增多。細(xì)胞核內(nèi)可見NICD表達(dá),以終末期最為明顯。4隨病程進(jìn)展,ALS小鼠腰髓Jagged-1陽性細(xì)胞進(jìn)行性增多,Jagged-1表達(dá)于小膠質(zhì)細(xì)胞。5隨病程進(jìn)展,ALS小鼠腰髓DLL4陽性神經(jīng)元減少,而DLL4陽性星形膠質(zhì)細(xì)胞進(jìn)行性增多。6 ALS小鼠腰髓Hes-1表達(dá)主要見于細(xì)胞核,隨病程進(jìn)展Hes-1陽性細(xì)胞進(jìn)行性增多。結(jié)論:1 SOD1-G93A轉(zhuǎn)基因小鼠腰髓存在少突膠質(zhì)細(xì)胞變性及少突膠質(zhì)前體細(xì)胞增生。2 SOD1-G93A轉(zhuǎn)基因小鼠腰髓激活的星形膠質(zhì)細(xì)胞表達(dá)ET-1。3 SOD1-G93A轉(zhuǎn)基因小鼠腰髓存在Notch1激活及其配體、靶基因的表達(dá)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 朱文麗;吳軍;;Notch信號通路在神經(jīng)系統(tǒng)疾病中的研究進(jìn)展[J];中國神經(jīng)免疫學(xué)和神經(jīng)病學(xué)雜志;2015年02期

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