發(fā)布時間：2017-12-27 20:05

  本文關(guān)鍵詞：鞘內(nèi)注射ssAAV9-DAO對ALS轉(zhuǎn)基因小鼠模型的保護作用及機制研究　出處：《河北醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肌萎縮側(cè)索硬化 D-氨基酸氧化酶 腺相關(guān)病毒 NF-κB Akt

【摘要】：肌萎縮側(cè)索硬化(Amyotrophic lateral sclerosis,ALS)是一種成人起病的、進行性、致死性神經(jīng)系統(tǒng)變性疾病,病變累及皮層、腦干及脊髓前角運動神經(jīng)元,導(dǎo)致全身肌肉的進行性萎縮、無力。患者多于發(fā)病后的3~5年內(nèi)因呼吸肌萎縮無力而死亡。由于其發(fā)病機制的復(fù)雜性,目前尚無有效的治療方法。ALS的患病率約為5~7/10萬,其中約90%為散發(fā)型肌萎縮側(cè)索硬化(SALS),其余10%為家族性肌萎縮側(cè)索硬化(FALS),約20%的FALS是由銅/鋅超氧化物歧化酶1基因(SOD1)的突變引起。SOD1-G93A轉(zhuǎn)基因小鼠為SOD1基因93號位點發(fā)生甘氨酸→精氨酸的突變,高度模擬人類ALS疾病的病理及臨床特點,被廣泛地應(yīng)用于ALS病理機制的研究。ALS的病理機制目前尚未完全明確,已知的有蛋白錯誤折疊、興奮性氨基酸毒性、線粒體損傷、氧化應(yīng)激等,近年來,一些新的證據(jù)表明D-絲氨酸(D-serin,D-Ser)—一種內(nèi)源性的N-甲基-D-天冬氨酸受體(NMDAR)的協(xié)同激動劑,在ALS病人中有所升高,并可加劇運動神經(jīng)元的死亡。而造成D-Ser升高的主要原因在于D-氨基酸氧化酶(D-amino acid oxidase DAO)的活性降低。DAO是一種以黃素腺嘌呤二核苷酸(FAD)為輔基的典型黃素蛋白酶類,可氧化D-氨基酸如D-丙氨酸、D-絲氨酸、D-脯氨酸的氨基生成相應(yīng)的酮酸。DAO廣泛存在于哺乳動物的中樞神經(jīng)系統(tǒng)(CNS)、肝臟及腎臟,在CNS主要存在于腦干和脊髓。在哺乳動物和低等生物如酵母、真菌和細菌的DAO蛋白之中,Arg199殘基緊靠FAD結(jié)合位點,處于Tyr228和His307殘基之間,對酶活性起著至關(guān)重要的作用。發(fā)生于此位點的突變(R199W)會在細胞中引起不良后果,可導(dǎo)致ALS的發(fā)生發(fā)展。研究認為,脊髓內(nèi)存在的高水平的DAO主要作用在于降解D-Ser,從而通過NMDAR介導(dǎo)的神經(jīng)毒性調(diào)控神經(jīng)元的存活。而不論在f ALS患者中還是在SOD1-G93A模型小鼠中,腰髓DAO水平均有所降低。我們設(shè)想補充ALS小鼠模型腰髓中的DAO水平可能會增加D-Ser的降解,從而減輕NMDAR過度激活造成的神經(jīng)毒性,保護運動神經(jīng)元。自1952年DNA被發(fā)現(xiàn)作為遺傳物質(zhì)以來,利用載體(如病毒)攜帶DNA以治療遺傳性疾病的方法便應(yīng)運而生。常用于轉(zhuǎn)導(dǎo)中樞神經(jīng)系統(tǒng)的載體有包含質(zhì)粒的納米顆粒、逆轉(zhuǎn)錄病毒重組載體、腺相關(guān)病毒(AAV)、腺病毒和單純皰疹病毒(HSV)。其中AAV已成為最安全、最常用的一種用于傳遞治療基因的病毒載體。本研究應(yīng)用CMV啟動子調(diào)控下的單鏈腺相關(guān)病毒血清9型(ss AAV9),攜帶編碼的小鼠DAO基因(ss AAV9-DAO)經(jīng)鞘內(nèi)注射的途徑干預(yù)發(fā)病早期的SOD1-G93A轉(zhuǎn)基因小鼠,觀察上調(diào)小鼠腰髓內(nèi)的DAO的水平對其行為學(xué)及病理表現(xiàn)的影響,并進一步探索其可能的機制。我們查閱文獻尚未見類似報道。第一部分ALS小鼠模型中DAO的表達及分布目的:我們對不同時期(60天齡—癥狀前期、90天齡—癥狀早期、120~140天齡—終末期)的SOD1-G93A轉(zhuǎn)基因小鼠腰髓組織內(nèi)的DAO水平進行檢測,觀察DAO水平隨病程進展發(fā)生的變化,并進一步觀察DAO所在的細胞類型,為病毒載體系統(tǒng)的選擇提供依據(jù)。方法:我們選取由美國Jackson實驗室提供的SOD1-G93A轉(zhuǎn)基因小鼠作為動物模型,并應(yīng)用其提供的引物序列和鑒定方法進行DNA鑒定,鑒定陽性的60天、90天及終末期小鼠作為研究對象,并以相同天齡的陰性小鼠作為陰性對照(即60天陽性、60天陰性、90天陽性、90天陰性、終末陽性、終末陰性共6組),每組4只小鼠。小鼠新鮮取材后采用蛋白免疫印跡法(Western blot)檢測小鼠腰髓DAO的表達水平。另取90d陽性小鼠3只,灌注取材后行25μm震蕩切片,采用免疫熒光共聚焦技術(shù)(confocal)觀察DAO主要存在的細胞類型。結(jié)果:1與陰性鼠比較,SOD1-G93A小鼠腰髓DAO蛋白水平在終末期顯著減少(P0.05),其他時期則無顯著變化。2免疫熒光共聚焦染色結(jié)果顯示SOD1-G93A小鼠腰髓DAO主要表達在運動神經(jīng)元,此外星形膠質(zhì)細胞和小膠質(zhì)細胞也有極少量的表達。結(jié)論:SOD1-G93A小鼠腰髓組織內(nèi)的DAO表達在終末期時顯著減少,說明了補充腰髓DAO可能對SOD1-G93A小鼠具有保護作用;SOD1-G93A小鼠腰髓中DAO主要存在于運動神經(jīng)元,這對選取神經(jīng)元靶向的載體系統(tǒng)提供了依據(jù)。第二部分鞘內(nèi)注射ss AAV9在SOD1-G93A轉(zhuǎn)基因小鼠脊髓轉(zhuǎn)導(dǎo)效率的研究目的:通過給SOD1-G93A轉(zhuǎn)基因小鼠鞘內(nèi)注射ss AAV9-GFP和ss AAV9-DAO,觀察在此種方法下,綠色熒光蛋白(GFP)在SOD1-G93A小鼠脊髓內(nèi)的分布情況及轉(zhuǎn)導(dǎo)效率,以及小鼠腰髓組織DAO及D-Ser表達的變化,以確定此種方法的可行性。方法:選取SOD1-G93A轉(zhuǎn)基因小鼠作為研究對象,單次鞘內(nèi)注射ss AAV9-GFP(1×1012vg/kg)或ss AAV9-DAO(1×1012vg/kg),給藥3周后部分小鼠以4%的多聚甲醛液灌注固定,行脊髓組織25μm震蕩切片,部分小鼠新鮮取材,分離腰髓組織。通過免疫熒光共聚焦技術(shù)觀察GFP表達及分布情況,蛋白免疫印跡法(Western blot)檢測小鼠腰髓DAO的表達水平,免疫組織化學(xué)染色法觀察小鼠腰髓D-Ser的表達。結(jié)果:1脊髓切片免疫熒光染色顯示腰髓可見較多GFP表達,另外骶髓、胸髓也有少許GFP表達,頸髓則很少見。2免疫熒光共聚焦染色顯示GFP與神經(jīng)元共定位,而不表達于星形膠質(zhì)細胞和小膠質(zhì)細胞,且SOD1-G93A轉(zhuǎn)基因小鼠腰髓運動神經(jīng)元轉(zhuǎn)導(dǎo)效率約為12%。3 Western blotting結(jié)果顯示ss AAV9-DAO組小鼠腰髓組織DAO表達明顯增多,同時免疫組織化學(xué)染色結(jié)果顯示,與ss AAV9-GFP組小鼠相比,ss AAV9-DAO組小鼠腰髓組織D-Ser陽性細胞數(shù)顯著減少。結(jié)論:GFP主要表達于注射點附近的脊髓部位,腰髓為主;GFP主要表達于神經(jīng)元,且腰髓運動神經(jīng)元轉(zhuǎn)導(dǎo)效率約為12%;ss AAV9-DAO單次鞘內(nèi)注射可增加SOD1-G93A小鼠腰髓內(nèi)的DAO表達,同時降低D-Ser水平。說明了此種給藥方式可補充小鼠腰髓內(nèi)的DAO水平并具有酶活性。第三部分鞘內(nèi)注射ss AAV9-DAO對SOD1-G93A轉(zhuǎn)基因小鼠行為學(xué)影響及機制研究目的:通過給SOD1-G93A轉(zhuǎn)基因小鼠鞘內(nèi)注射ss AAV9-DAO,觀察過表達DAO對SOD1-G93A小鼠生存期、體重、轉(zhuǎn)輪試驗及組織病理改變的影響,并探索相關(guān)的保護機制。方法:選取由美國Jackson實驗室提供的SOD1-G93A轉(zhuǎn)基因小鼠作為ALS動物模型,并應(yīng)用其提供的引物序列和鑒定方法進行DNA鑒定,選擇同窩配對的陽性鼠作為受試對象。小鼠90天齡時,選取體重接近(組間平均起始體重差別≤0.3g)的同窩小鼠,雌、雄各15對,隨機分配到治療組和對照組,分別給予ss AAV9-DAO和ss AAV9-GFP鞘內(nèi)注射,觀察小鼠體重、轉(zhuǎn)輪變化及生存期。另外選取給予同樣干預(yù)方法的小鼠進行機制研究,給藥3周后取材,其中3對用0.05%的戊二醛-多聚甲醛液灌注固定,腰髓25μm震蕩切片,通過免疫組織化學(xué)染色法觀察SMI-32、Iba-1、GFAP的改變;L5前根切片甲苯胺藍染色觀察軸索破壞情況;余4對新鮮取材,腓腸肌冰凍切片HE染色觀察肌纖維情況;通過蛋白免疫印跡法檢測腰髓TNF-α、IKBα、IKKβ、IKKγ、p-P65、p-Akt、Bcl-2、Bax、Caspase3、Caspase9、Cd11b、GFAP的水平。結(jié)果:1與ss AAV9-GFP組小鼠相比,ss AAV9-DAO組小鼠生存期延長6天,差異有統(tǒng)計學(xué)意義(n=30,P0.05),其中,雌鼠延長8天,差異顯著(n=15,P0.05),雄鼠也延長8天,但差異無統(tǒng)計學(xué)意義(n=15,P0.05)。雄鼠兩組間的體重、轉(zhuǎn)輪均無顯著差異;雌鼠ss AAV9-DAO組僅在110天齡時短暫改善轉(zhuǎn)輪表現(xiàn)(P0.05),體重則無顯著差異。2免疫組織化學(xué)染色結(jié)果顯示,與ss AAV9-GFP組小鼠相比,ss AA V9-DAO組小鼠腰髓組織SMI32表達增多、GFAP、Iba-1表達減少,同時Western blotting結(jié)果顯示,ss AAV9-DAO組小鼠腰髓組織Cd11b、GFAP表達較對照組減少。即治療組腰髓運動神經(jīng)元保留較對照組明顯增多,而星形膠質(zhì)細胞、小膠質(zhì)細胞增生較對照組明顯減少。3 L5前根切片甲苯胺藍染色結(jié)果顯示,與對照組小鼠相比,給藥組小鼠L5前根保留的軸索數(shù)量明顯增多。腓腸肌冰凍切片HE染色結(jié)果顯示,對照組小鼠肌纖維大小不一,可見大量中央核,給藥組較之有所改善。4腰髓組織Western blotting結(jié)果顯示,與ss AAV9-GFP組相比,ss AAV9-DAO組的NF-κB信號通路正向調(diào)節(jié)因子IKKβ、IKKγ水平顯著降低,而負向調(diào)節(jié)因子IKBα顯著增高,另外反映NF-κB信號通路激活狀態(tài)的p-P65、TNF-α水平均顯著降低(P0.05)。同時,ss AAV9-DAO組促凋亡因子Bax、Caspase3、Caspase9均顯著降低,而抗凋亡因子Bcl-2顯著增高,且伴有p-Akt表達的增高(P0.05)。結(jié)論:ss AAV9-DAO單次鞘內(nèi)注射可延長癥狀期SOD1-G93A小鼠的生存期,但存在性別差異,造成此種差異的原因尚不完全明確。但對體重、轉(zhuǎn)輪表現(xiàn)的改善不顯著;ss AAV9-DAO可減少SOD1-G93A小鼠腰髓運動神經(jīng)元的丟失和小膠質(zhì)細胞、星形膠質(zhì)細胞的增生活化,并對前根的軸索及肌肉纖維也具有一定保護作用;DAO的保護作用可能與其抑制NF-κB的激活從而抑制炎癥反應(yīng),抑制Akt的失磷酸化從而減少凋亡反應(yīng)有關(guān)。確切的機制尚有待進一步研究。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is an adult onset, progressive and fatal neurodegenerative disease. Lesions involve motor neurons in the cortex, brain stem and spinal cord, resulting in progressive atrophy and weakness of the whole muscle. The patients died of respiratory muscle atrophy in 3~5 years after the onset of the disease. Because of the complexity of its pathogenesis, there is no effective treatment at present. The prevalence of ALS is about 5~7/10 million, about 90% of which are sporadic amyotrophic lateral sclerosis (SALS), and the rest 10% are familial amyotrophic lateral sclerosis (FALS). About 20% of FALS is caused by mutations in Cu / Zn superoxide dismutase 1 gene (SOD1). SOD1-G93A transgenic mice have been genetically modified by glycine to arginine at site 93 of SOD1 gene, which is highly simulating the pathological and clinical characteristics of human ALS disease. It is widely used in ALS pathological mechanism research. The pathogenesis of ALS is not yet entirely clear, known to have protein misfolding, excitotoxicity, mitochondrial damage, oxidative stress, in recent years, some new evidence that D- serine (D-serin, D-Ser) - N- methyl -D- aspartate receptor (NMDAR) is a kind of endogenous CO agonist, has increased in ALS patients, and can aggravate the death of motor neurons. The main reason for the increase of D-Ser is that the activity of D- amino acid oxidase (D-amino acid oxidase DAO) is reduced. DAO is a typical flavin protease that uses flavin adenine dinucleotide (FAD) as a complementary base, and it can oxidize amino acids such as D- alanine, D- serine and D- proline to produce corresponding keto acids. D- can also be used to produce ketoacids. DAO exists widely in the central nervous system (CNS), liver and kidney of mammals. In CNS, it mainly exists in the brain stem and spinal cord. In mammalian and lower organisms, such as yeast, fungi and bacteria, the Arg199 residues are closely related to FAD binding sites, which are between Tyr228 and His307 residues, and play an important role in DAO activity. The mutation (R199W) that occurs at this site can cause adverse effects in the cell, which can lead to the development of ALS. It is believed that the major role of the high level of DAO in the spinal cord is to degrade D-Ser, which regulates the survival of neurons through NMDAR mediated neurotoxicity. Both in the f ALS patients and in the SOD1-G93A model mice, the DAO level of the lumbar spinal cord decreased. We assume that the DAO level in the lumbar spinal cord of the supplementary ALS mouse model may increase the degradation of D-Ser, thereby reducing the neurotoxicity caused by excessive activation of NMDAR and protecting the motor neurons. Since the discovery of DNA as a genetic material in 1952, the use of carriers (such as viruses) to carry DNA for the treatment of hereditary diseases has emerged. Vectors commonly used to transduce the central nervous system include nanoparticles containing plasmid, retroviral recombinant vectors, adeno-associated virus (AAV), adenovirus and herpes simplex virus (HSV). AAV has become the safest and most commonly used virus carrier for the transmission of therapeutic genes. The research and application of CMV promoter single stranded adeno-associated virus type 9 serum sub regulation (SS AAV9), mice carrying the DAO gene encoding (SS AAV9-DAO) by intrathecal injection of the interventions in the early onset of SOD1-G93A transgenic mice, to observe the effect of up regulation of spinal cord of mice in the DAO level of the behavior and pathological findings, and to explore its possible mechanism. We have not found similar reports in the literature. The first part of the expression and distribution of ALS in a mouse model of DAO Objective: we in different periods (60 days of age and symptoms early, at the age of 90 days and 120~140 days of age, early symptoms and ESRD) were detected in SOD1-G93A transgenic mouse spinal cord in the DAO level, to observe the change of DAO level with the development of course of disease occurrence. And to observe DAO's cell types, provide the basis for the selection of virus vector system. Methods: We selected SOD1-G93A transgenic mice provided by the United States Jackson laboratory as an animal model, and use of the primer sequences and identification methods of DNA identification, identification of positive 60 days, 90 days and end-stage mice as the research object, and on the same day old mice were used as negative control (i.e., positive for 60 days 60 days, 90 days, negative positive negative and positive at the end of 90 days, a total of 6 terminal negative group), 4 mice in each group. Western blot was used to detect the expression level of DAO in the lumbar spinal cord of mice. In addition, 3 90d positive mice were taken, and 25 mu m was sliced after perfusion. The main cell types of DAO were observed by immunofluorescence confocal technique (confocal). Results: 1 compared with the negative mice, the level of DAO protein in the lumbar spinal cord of SOD1-G93A mice decreased significantly at the end stage (P0.05), but there was no significant change at other times. 2 immunofluorescence confocal staining showed that DAO in lumbar spinal cord of SOD1-G93A mice was mainly expressed in motoneurons, and astrocytes and microglia also had very few expression. Conclusion: SOD1-G93A mice spinal cord tissue expression of DAO decreased significantly in end-stage, that of lumbar spinal DAO may have protective effects on SOD1-G93A mice; SOD1-G93A mice spinal cord in DAO are mainly in the selection of motor neurons, neuron targeting carrier system provides a basis for. The second part of intrathecal injection of SS AAV9 in the study of SOD1-G93A transgenic mouse spinal cord transduction efficiency to SOD1-G93A transgenic mice of intrathecal injection of SS AAV9-GFP and SS AAV9-DAO, were observed in this way, the green fluorescent protein (GFP) distribution and transduction efficiency in SOD1-G93A mouse spinal cord, and the expression of DAO and D-Ser in mice spinal cord tissue, to determine the feasibility of this method. Methods: SOD1-G93A transgenic mice were selected as subjects. Single dose intrathecal injection of SS AAV9-GFP (1 x 1012vg/kg) or SS AAV9-DAO (1 x 1012vg/kg) was administered for 3 weeks, and some mice were 4% polypeptides.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R744.8;R-332

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