發(fā)布時間：2018-04-23 08:30

  本文選題：帕金森病 + α-synuclein��； 參考：《北京理工大學(xué)》2015年博士論文

【摘要】：帕金森病(Parkinson’s disease,PD)是目前第二大神經(jīng)退行性疾病,多于老年發(fā)病,其癥狀表現(xiàn)為靜止性震顫,身心反應(yīng)遲鈍以及強直等,對生活質(zhì)量造成嚴(yán)重的影響,且中國PD病人數(shù)量呈逐年增長的趨勢。PD的兩大病理特征為黑質(zhì)區(qū)多巴胺能神經(jīng)元的喪失與蛋白聚集體——路易小體(Lewy Bodies,LBs)的形成。關(guān)于PD的發(fā)病有很多假說,例如氧化應(yīng)激假說,自由基假說等,然而其發(fā)病機制目前尚不清楚。遺傳因素和環(huán)境因素被認(rèn)為是PD的兩個主要的致病原因,其中環(huán)境因素起主導(dǎo)作用。自從MPTP被發(fā)現(xiàn)以來,神經(jīng)毒素作為PD的致病因子受到了廣泛關(guān)注,兒茶酚四氫異喹啉類化合物(catechol tetrahydroisoquinolines,CTIQs)就是其中一類內(nèi)源性的神經(jīng)毒素。本課題組針對CTIQs在體內(nèi)形成與代謝進(jìn)行了長期研究,提出了CTIQs類神經(jīng)毒素腦內(nèi)形成與神經(jīng)毒性的假說。當(dāng)腦受到外界刺激(物理打擊或環(huán)境污染等)會產(chǎn)生氧化應(yīng)激導(dǎo)致脂質(zhì)過氧化(lipid peroxidation,LPO),而LPO的終產(chǎn)物是醛類,醛類與多巴胺(dopamine,DA)反應(yīng)后生成Salsolinol(Sal)等CTIQs,CTIQs經(jīng)過氮甲基化和氧化代謝產(chǎn)生能夠與線粒體復(fù)合體Ⅰ結(jié)合的離子化合物,造成細(xì)胞能量代謝障礙,進(jìn)一步加劇氧化應(yīng)激。另外,形成的CTIQs有可能促進(jìn)腦內(nèi)α-synuclein聚集的發(fā)生,而聚集進(jìn)一步產(chǎn)生氧化應(yīng)激,從而形成循環(huán),其結(jié)果使神經(jīng)元變性死亡。在本課題中,針對以上假說,通過構(gòu)建α-synuclein轉(zhuǎn)基因大鼠模型,重點研究內(nèi)源性神經(jīng)毒素N-methyl-Salsolinol(NMSal)在腦內(nèi)對α-synuclein聚集的影響,闡明α-synuclein腦內(nèi)聚集的機制,考察NMSal的神經(jīng)毒性。通過本文的研究,對上述假說提供實驗數(shù)據(jù)積累。本研究取得了以下成果:1、成功建立了腦內(nèi)過表達(dá)α-synuclein的大鼠PD模型。采用了重組腺相關(guān)病毒(Recombinant Adeno-Associated Viral,rAAV)作為基因表達(dá)的載體,在研究過程中建立了一個簡單,高效的純化rAAV的方法,最終得到了滴度超過1013gc/mL的rAAV。利用腦立體定位技術(shù)將rAAV注射至大鼠大腦黑質(zhì)致密部后,經(jīng)過一系列時間節(jié)點的行為學(xué)觀察,α-synuclein WT組和A53T組都會引發(fā)針對單側(cè)多巴胺能神經(jīng)元損傷的行為——阿撲嗎啡誘導(dǎo)向健側(cè)轉(zhuǎn)圈,其中α-synuclein A53T組轉(zhuǎn)圈速度為3.91±1.62圈/min,而WT組速度為2.68±1.74圈/min。對其腦部切片后進(jìn)行免疫組化檢測,外源性α-synuclein特異性地在多巴胺能神經(jīng)元中表達(dá),其中α-synuclein WT組從第3周至第12周,表達(dá)程度呈逐漸增加的趨勢,而α-synucleina53t組至第9周時表達(dá)已至頂峰。同時對多巴胺能神經(jīng)元標(biāo)志性物質(zhì)——酪氨酸羥化酶(tyrosinehydroxylase,th)的免疫組化檢測表明,多巴胺能神經(jīng)元的死亡與α-synuclein表達(dá)程度呈現(xiàn)正相關(guān)的趨勢。以上結(jié)果說明α-synucleinwt與α-synucleina53t兩種大鼠pd模型建立成功。2、nmsal可增強大鼠pd模型中α-synuclein產(chǎn)生的神經(jīng)毒性。在成功建立大鼠pd模型的基礎(chǔ)上,再次通過立體定位手術(shù)在其黑質(zhì)區(qū)注射100nmolnmsal,誘導(dǎo)4周之后,其行為學(xué)指標(biāo)顯示nmsal的誘導(dǎo)可增加α-synucleinwt組與a53t組中阿撲嗎啡誘導(dǎo)轉(zhuǎn)圈速度。對其黑質(zhì)紋狀體系統(tǒng)進(jìn)行免疫組化檢測表明,nmsal能夠促進(jìn)大鼠pd模型中多巴胺能神經(jīng)元的喪失,且α-synucleinwt組和a53t組的凋亡率分別上升了3.7%和7%。以上的結(jié)果顯示nmsal能夠引發(fā)大鼠pd模型中多巴胺能系統(tǒng)在原來基礎(chǔ)上受到更嚴(yán)重的損傷。使用elisa的方法測定紋狀體區(qū)丙二醛(malondialdehyde,mda)和活性氧基團(tuán)(reactiveoxygenspecies,ros)的結(jié)果顯示nmsal在α-synucleina53t組能夠?qū)⑦@兩個參數(shù)值提高50%,證明nmsal能夠在α-synuclein過表達(dá)的鼠腦中促進(jìn)氧化應(yīng)激的發(fā)生。伴隨著多巴胺能神經(jīng)元的喪失,紋狀體區(qū)多巴胺的含量也隨之降低,其中α-synuclein組wt組和a53t組da含量分別約為對照組的44%與27%,nmsal的誘導(dǎo)可以進(jìn)一步降低da含量,在wt組和a53t組分別僅為對照組的24%與15%。而內(nèi)源性神經(jīng)毒素的測定結(jié)果則證實了nmsal誘導(dǎo)之后對大鼠pd模型腦內(nèi)sal和nmsal的生成具有促進(jìn)作用。這些結(jié)果表明nmsal可以在大鼠pd模型中促進(jìn)α-synuclein蛋白毒性作用的發(fā)揮,并且其促進(jìn)效果在突變體的α-synucleina53t組表現(xiàn)更為顯著。3、nmsal促進(jìn)鼠腦中類似lbs的α-synuclein蛋白聚集。使用免疫印跡檢測紋狀體中不溶性的組分,結(jié)果顯示經(jīng)過nmsal誘導(dǎo)之后,α-synuclein組wt組和a53t組中α-synuclein三聚體的量分別增加為原來的2倍與3.5倍,免疫組化的結(jié)果顯示nmsal的誘導(dǎo)會在紋狀體中增加抵抗蛋白酶k消化的α-synuclein顆粒的數(shù)目。以上結(jié)果說明nmsal會促進(jìn)腦內(nèi)α-synuclein聚集的發(fā)生。使用s129磷酸化的α-synuclein抗體對黑質(zhì)和紋狀體區(qū)進(jìn)行免疫組化染色的結(jié)果說明,只有在α-synucleina53t組才有顯著的s129磷酸化發(fā)生,在紋狀體區(qū)可以看到散布的顆粒存在,統(tǒng)計結(jié)果顯示nmsal誘導(dǎo)前后顆粒數(shù)目分別為4.5±1.7和8.8±3.3,這說明nmsal可促進(jìn)α-synuclein磷酸化的發(fā)生。而對紋狀體進(jìn)行泛素和α-synuclein免疫熒光染色的結(jié)果顯示,在紋狀體區(qū)出現(xiàn)了泛素與α-synuclein的共定位,說明出現(xiàn)了α-synuclein的泛素化,對熒光強度進(jìn)行定量后可知,NMSal誘導(dǎo)可增加α-synuclein的泛素化程度。在紋狀體中出現(xiàn)的顆粒具有磷酸化與泛素化的特性說明此α-synuclein聚集的顆粒具有與LBs相類似的性質(zhì)。由此可知,NMSal可以促進(jìn)α-synuclein聚集的發(fā)生,并產(chǎn)生類似LBs的蛋白聚集顆粒,且NMSal對α-synuclein A53T組的促進(jìn)作用更為明顯。4、NMSal可能抑制α-synuclein降解通路中的自噬-溶酶體途徑。使用免疫印跡的方法檢測了α-synuclein兩大降解通路:自噬-溶酶體途徑和泛素蛋白酶體系統(tǒng),結(jié)果顯示,在NMSal誘導(dǎo)之后,可導(dǎo)致大鼠PD模型鼠腦中自噬-溶酶體途徑中Beclin-1、Atg5和Atg3的下調(diào),而對Parkin和UCHL1的免疫印跡結(jié)果則顯示NMSal的誘導(dǎo)對泛素蛋白酶體途徑?jīng)]有顯著性的影響。此結(jié)果表明NMSal引發(fā)α-synuclein聚集的過程中一個可能的機制是阻斷其降解通路中的自噬-溶酶體途徑。本課題通過立體定位手術(shù)在大鼠PD模型中建立了α-synuclein過表達(dá)與NMSal直接定位誘導(dǎo)的實驗,首次在動物腦內(nèi)研究了過量的NMSal和過表達(dá)α-synuclein的直接相互作用,得出了NMSal可促進(jìn)過表達(dá)的α-synuclein聚集并增強其對多巴胺能神經(jīng)元的毒性的結(jié)論。這些結(jié)果為CTIQs類神經(jīng)毒素腦內(nèi)形成與神經(jīng)毒性的假說的證明提供了體內(nèi)的實驗證據(jù),并且為假說的進(jìn)一步研究提供了非常有用的動物模型。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is the second major neurodegenerative disease, more than the elderly, its symptoms are static tremor, mental retardation and ankylosis, which have a serious impact on the quality of life, and the number of PD patients in China is increasing year by year and the two major pathological characteristics of.PD are dopamine in the substantia nigra area. The loss of neurons and protein aggregates - the formation of the Lewy Bodies (Lewy Bodies, LBs). There are many hypotheses about the pathogenesis of PD, such as the oxidative stress hypothesis, the free radical hypothesis, etc. However, the pathogenesis is not yet clear. Genetic and environmental factors are considered to be the two main causes of PD, of which environmental factors play a leading role. Since MPTP was discovered, neurotoxin has been widely concerned as a pathogenic factor of PD. The catechol tetrahydroisoquinolines (CTIQs) of catechol (catechol tetrahydroisoquinolines, CTIQs) is a kind of endogenous neurotoxin. This research group has studied the form and metabolism of CTIQs in the body for a long time, and proposed the CTIQs class God. The hypothesis that the brain is formed and neurotoxic through the toxin. When the brain is stimulated by external stimuli (physical shock or environmental pollution), oxidative stress leads to lipid peroxidation (lipid peroxidation, LPO), and the final products of LPO are aldehydes, aldehydes and dopamine (dopamine, DA) react to Salsolinol (Sal) and other CTIQs, CTIQs through nitrogen methylation and oxidation. Metabolism produces ionic compounds that combine with mitochondrial complex I, causing cell energy metabolic disorders and further aggravating oxidative stress. In addition, the formation of CTIQs may promote the occurrence of alpha -synuclein aggregation in the brain, and the aggregation further produces oxidative stress and forms a cycle, which results in degeneration of neurons. In view of the above hypothesis, the effect of endogenous neurotoxin N-methyl-Salsolinol (NMSal) on the aggregation of alpha -synuclein in the brain was studied by constructing a -synuclein transgenic rat model. The mechanism of alpha -synuclein accumulation in the brain was elucidated and the neurotoxicity of NMSal was investigated. The experimental data were provided through this study. The following achievements have been obtained: 1, a rat model of PD was successfully established in the brain that overexpressed alpha -synuclein. The recombinant adeno-associated virus (Recombinant Adeno-Associated Viral, rAAV) was used as a carrier of gene expression. A simple, efficient method for purifying rAAV was established in the study, and the titer exceeded 1013gc/mL in the end. After injection of rAAV into the dense part of the substantia nigra of the rat brain by using rAAV. stereotaxic technique, the behavior of a series of time nodes was observed. Both the alpha -synuclein WT group and the A53T group would cause the behavior of the unilateral dopaminergic neuron injury - apomorphine induced Xiang Jian's side circle, of which the speed of the alpha -synuclein A53T group was 3.. 91 + 1.62 laps /min, while group WT was 2.68 + 1.74 laps /min. to detect the brain slices with immunohistochemical staining. Exogenous alpha -synuclein was specifically expressed in dopaminergic neurons. The expression level of alpha -synuclein WT group increased gradually from third to twelfth weeks, and the expression of alpha -synucleina53t group was at the top of ninth weeks. At the same time, the immunohistochemical detection of the Tyrosinehydroxylase (th), a marker of dopaminergic neurons, showed that the death of dopaminergic neurons was positively correlated with the expression of alpha -synuclein. The above results showed that the PD model of two rats of alpha -synucleinwt and alpha -synucleina53t was successful in establishing.2, nmsal. The neurotoxicity induced by alpha -synuclein in the rat PD model was enhanced. On the basis of the successful establishment of the rat PD model, 100nmolnmsal was injected into the substantia nigra by stereotaxic operation again. After 4 weeks of induction, its behavioral indicators showed that nmsal induction increased the speed of the alpha -synucleinwt group and the induced rotation of morphine in the a53t group. The immunohistochemical detection of the nigrostriatal system showed that nmsal could promote the loss of dopaminergic neurons in the PD model of rats, and the apoptosis rate of the alpha -synucleinwt group and the a53t group increased by 3.7% and 7%. respectively. The results showed that nmsal could cause the dopaminergic system in the rat PD model to be more severely damaged on the basis of the original basis. The results of the determination of malondialdehyde, MDA and reactive oxygen groups (reactiveoxygenspecies, ROS) in the striatum method by ELISA show that nmsal can increase these two parameters by 50% in the group of alpha -synucleina53t, proving that nmsal can promote oxidative stress in the rat brain of alpha -synuclein overexpression. The content of dopamine in the striatum decreased with the loss of the element. The Da content of group WT and a53t in group a -synuclein group was about 44% and 27% in the control group, respectively. The induction of nmsal could further reduce the content of Da. In WT group and a53t group, only 24% and 15%. in the control group and the results of endogenous neurotoxin test confirmed nmsal induction. The results showed that Sal and nmsal were promoted in the brain of rat PD model. These results showed that nmsal could promote the toxicity of alpha -synuclein protein in the rat PD model, and its promoting effect was more significant in the alpha -synucleina53t group of the mutant, and nmsal promoted the aggregation of alpha -synuclein protein in the rat brain similar to lbs. The insoluble fractions in the striatum were detected by immunoblotting. The results showed that after nmsal induction, the amount of alpha -synuclein trimer in group WT and a53t group increased to 2 times and 3.5 times, respectively. The results of immunohistochemistry showed that the induction of nmsal could be added to the alpha -synuclein particles digested by protease K in the striatum. The above results indicate that nmsal promotes the occurrence of alpha -synuclein aggregation in the brain. The results of immunohistochemical staining of substantia nigra and striatum by s129 phosphorylated alpha -synuclein antibody show that only a significant s129 phosphorylation occurs in the alpha -synucleina53t group, and the scattered particles can be found in the striate region and the statistical junction is observed. The results showed that the number of nmsal particles was 4.5 + 1.7 and 8.8 + 3.3 before and after induction, indicating that nmsal could promote the occurrence of alpha -synuclein phosphorylation. The results of ubiquitin and alpha -synuclein immunofluorescence staining in the striatum showed that ubiquitin and alpha -synuclein were found in the striatum, indicating the presence of ubiquitination of alpha -synuclein. When the fluorescence intensity is quantified, NMSal induction can increase the degree of ubiquitination of alpha -synuclein. The particles in the striatum have the properties of phosphorylation and ubiquitination indicating that the particles of this -synuclein aggregation are similar to that of LBs. Thus, NMSal can promote the occurrence of alpha -synuclein aggregation and produce a similar LBs. Protein aggregation particles, and the promotion of NMSal to the -synuclein A53T group more obviously.4, NMSal may inhibit the autophagy lysosome pathway in the alpha -synuclein degradation pathway. Using the immunoblotting method to detect the large degradation pathway of alpha -synuclein two: autophagy lysosome pathway and ubiquitin proteasome system. The results show that NMSal induced by NMSal After that, it could lead to the downregulation of Beclin-1, Atg5 and Atg3 in the autophagy lysosome pathway of rat PD model, while the immunoblotting results of Parkin and UCHL1 showed that the induction of NMSal had no significant effect on the ubiquitin proteasome pathway. The results showed that a possible mechanism in the process of NMSal induced alpha -synuclein aggregation was to block its descending. The autophagy lysosome pathway in the pathway was solved by stereotaxic surgery in the rat PD model. The experiment was established to induce the over expression of alpha -synuclein and direct localization of NMSal in the rat model. The direct interaction of excessive NMSal and over expression of alpha -synuclein was first studied in the animal brain, and the NMSal could promote the over expressed alpha -synuclein aggregation. These results provide experimental evidence for the hypothesis of the brain formation and neurotoxicity of the CTIQs neurotoxin, and provide a very useful animal model for further research on the hypothesis.

【學(xué)位授予單位】：北京理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R742.5

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