本文選題：二苯乙烯苷 + α-突觸核蛋白　； 參考：《首都醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的α-突觸核蛋白(a-synuclein,α-syn)在多種神經(jīng)退行性疾病的發(fā)病機(jī)制中起著重要作用,這些疾病包括帕金森病、路易小體癡呆、阿爾茨海默病、多系統(tǒng)萎縮等,又統(tǒng)稱為突觸核蛋白病。聚集的α-Syn是路易小體的重要組成部分,而且突變的α-syn或多倍體α-syn可導(dǎo)致家族性帕金森病。但目前這些疾病都缺乏有效的治療藥物。二苯乙烯苷(2,3,5,4’-tetrahydroxy-stilbene glucoside,TSG)是中藥何首烏的主要有效成分和標(biāo)志成分。我們的前期研究發(fā)現(xiàn)TSG能夠抑制APP轉(zhuǎn)基因阿爾茨海默病小鼠模型海馬和大腦皮層α-syn過(guò)表達(dá)和聚集,抑制老年小鼠紋狀體和海馬α-syn過(guò)表達(dá)和聚集。本研究的目的是在A53T突變型α-syn轉(zhuǎn)基因小鼠模型和α-syn基因轉(zhuǎn)染神經(jīng)細(xì)胞模型上,進(jìn)一步研究TSG對(duì)α-syn過(guò)表達(dá)和聚集的影響及其在翻譯后修飾和蛋白降解方面的作用機(jī)制。方法1.應(yīng)用6月齡A53T突變型α-syn轉(zhuǎn)基因小鼠,灌胃給予TSG 6個(gè)月或9個(gè)月。應(yīng)用爬桿試驗(yàn)和筑巢試驗(yàn)檢測(cè)小鼠的運(yùn)動(dòng)能力,Y迷宮試驗(yàn)檢測(cè)小鼠的記憶能力;應(yīng)用免疫組化方法觀察小鼠腦內(nèi)酪氨酸羥化酶(TH)標(biāo)記的DA能神經(jīng)元、Iba-1標(biāo)記的小膠質(zhì)細(xì)胞;用Western blot法檢測(cè)小鼠腦中α-syn單體和寡聚體、磷酸化a-syn、Parkin、Beclin1、Atg、LC3的表達(dá)。2.采用α-syn基因轉(zhuǎn)染的SH-SY5Y細(xì)胞,與TSG孵育24 h后,換含小劑量魚藤酮的培養(yǎng)液繼續(xù)孵育24 h。應(yīng)用CCK8檢測(cè)細(xì)胞活性;免疫熒光雙標(biāo)法檢測(cè)α-syn的表達(dá);Western blot法檢測(cè)α-syn、磷酸化α-syn、磷酸化PP2A的表達(dá)。結(jié)果1.12月齡和15月齡a53tα-syn轉(zhuǎn)基因小鼠在爬桿試驗(yàn)中的潛伏期延長(zhǎng),筑巢行為評(píng)分減低,y迷宮自發(fā)性交替反應(yīng)減低;tsg灌胃給藥6個(gè)月或9個(gè)月能夠明顯縮短12月齡和15月齡模型小鼠在爬桿試驗(yàn)中的潛伏期,增高筑巢行為評(píng)分和y迷宮自發(fā)性交替反應(yīng),表明tsg能夠改善α-syn轉(zhuǎn)基因小鼠的運(yùn)動(dòng)功能和記憶能力。2.15月齡a53tα-syn轉(zhuǎn)基因模型組小鼠黑質(zhì)致密部th標(biāo)記的da能神經(jīng)元數(shù)量減少,在黑質(zhì)和紋狀體部位iba-1標(biāo)記的小膠質(zhì)細(xì)胞數(shù)量增高。tsg灌胃給藥9個(gè)月能夠增加模型小鼠黑質(zhì)致密部da能神經(jīng)元數(shù)量,減少黑質(zhì)和紋狀體部位小膠質(zhì)細(xì)胞數(shù)量,表明tsg具有神經(jīng)保護(hù)作用。3.15月齡a53tα-syn轉(zhuǎn)基因小鼠大腦皮層α-syn單體和寡聚體的表達(dá)增高;tsg能夠減低模型小鼠大腦皮層α-syn單體和寡聚體的表達(dá)水平,表明tsg對(duì)α-syn轉(zhuǎn)基因小鼠腦內(nèi)α-syn的過(guò)表達(dá)和聚集有抑制作用。4.在作用機(jī)制方面,我們發(fā)現(xiàn)15月齡a53tα-syn轉(zhuǎn)基因模型組小鼠紋狀體、大腦皮層中的α-syn在ser129位點(diǎn)的磷酸化水平增高;tsg能夠降低模型小鼠紋狀體和大腦皮層中α-syn的磷酸化水平(ser129位點(diǎn)),提示tsg能夠改善α-syn的翻譯后修飾,這可能是tsg抑制α-syn聚集的作用機(jī)制之一。5.tsg能夠增高15月齡a53tα-syn轉(zhuǎn)基因小鼠大腦皮層及小腦中parkin蛋白、atg5和lc3表達(dá);提示tsg可能通過(guò)上調(diào)泛素-蛋白酶體系統(tǒng)(usp)和自噬途徑從而增強(qiáng)α-syn的降解,這可能是tsg抑制α-syn過(guò)表達(dá)和聚集的另一個(gè)作用機(jī)制。6、在體外實(shí)驗(yàn)中,tsg能夠抑制α-syn基因轉(zhuǎn)染sh-sy5y細(xì)胞內(nèi)α-syn過(guò)表達(dá),降低魚藤酮復(fù)合α-syn基因轉(zhuǎn)染細(xì)胞模型中α-syn在ser129位點(diǎn)的磷酸化水平和pp2a的磷酸化水平,提示tsg可能通過(guò)下調(diào)pp2a的磷酸化引起pp2a活性增高,這可能是TSG抑制α-syn磷酸化的作用機(jī)制之一。結(jié)論TSG灌胃給藥能夠改善15月齡A53Tα-syn轉(zhuǎn)基因小鼠的運(yùn)動(dòng)能力和記憶功能,增高黑質(zhì)多巴胺能神經(jīng)元數(shù)量,抑制黑質(zhì)和紋狀體小膠質(zhì)細(xì)胞激活,抑制腦內(nèi)α-syn過(guò)表達(dá)和聚集;其作用機(jī)制可能涉及通過(guò)下調(diào)PP2A磷酸化而抑制α-syn的磷酸化,通過(guò)上調(diào)泛素-蛋白酶體系統(tǒng)和自噬途徑而增強(qiáng)α-syn的降解。結(jié)果提示TSG可能有利于防治突觸核蛋白相關(guān)的神經(jīng)退行性疾病。
[Abstract]:Objective alpha synuclein (a-synuclein, alpha -syn) plays an important role in the pathogenesis of a variety of neurodegenerative diseases. These diseases include Parkinson's disease, Louis's dementia, Alzheimer's disease, multi system atrophy and so on. They are also known as synuclein. The aggregated alpha -Syn is an important part of the Louis corpuscle, and the mutation is alpha. -syn or polyploid alpha -syn can lead to familial Parkinson's disease. But these diseases are currently lacking in effective therapeutic drugs. Two 2,3,5,4 '-tetrahydroxy-stilbene glucoside (TSG) is the main active component and marker of Polygonum multiflorum. Our preliminary study showed that TSG can inhibit APP transgenic Alzheimer's disease. The overexpression and aggregation of alpha -syn in the hippocampus and cerebral cortex of the rat model inhibit the overexpression and accumulation of alpha -syn in the striatum and hippocampus of the aged mice. The purpose of this study was to further study the effect of TSG on the over expression and aggregation of alpha -syn in the A53T mutated alpha -syn transgenic mouse model and the transfection of the alpha -syn gene into the neural cell model and the posttranslational repair. The mechanism of role of ornamentation and protein degradation. Method 1. using 6 month old A53T mutated alpha -syn transgenic mice, gavage was given to TSG for 6 months or 9 months. The exercise ability of mice was detected by climbing pole test and nesting test, the memory ability of mice was detected by Y maze test, and the immunohistochemical method was used to observe the tyrosine hydroxylase (TH) mark in the brain of mice. The DA neurons and Iba-1 labeled microglia were recorded, and the Western blot method was used to detect the alpha -syn monomer and oligomer in the brain of mice, phosphorylated a-syn, Parkin, Beclin1, Atg, LC3, and.2. using alpha -syn gene transfected cells. After incubating 24, the cells were incubated with small dose of rotenone and continued to incubate 24 applied detection cells. The expression of alpha -syn was detected by immunofluorescence double labeling; Western blot method was used to detect the expression of alpha -syn, phosphorylated alpha -syn, and phosphorylated PP2A. Results the incubation period of 1.12 month old and 15 month old a53t alpha -syn transgenic mice was prolonged in the climbing pole test, the score of nesting behavior was reduced, the spontaneous alternating response to the Y maze decreased, and TSG was administered for 6 months or 9 months by TSG. It can significantly shorten the incubation period of 12 month old and 15 month old model mice in the climbing pole test, increase the nest behavior score and the spontaneous alternating reaction of Y maze, which indicates that TSG can improve the motor function and memory ability of the -syn transgenic mice with.2.15 month old a53t alpha -syn transgenic model group, and the number of Da energy neurons marked in the dense part of the substantia nigra of the -syn transgenic mice Decrease, the increased number of microglia labeled by Iba-1 in substantia nigra and striatum.Tsg can increase the number of DA neurons in the dense part of the substantia nigra and decrease the number of microglia in the substantia nigra and striatum of the model mice for 9 months, indicating that TSG has neuroprotective effect on the cerebral cortex alpha -syn of.3.15 month old a53t alpha -syn transgenic mice. The expression of monomers and oligomers increased; TSG could reduce the expression level of alpha -syn monomers and oligomers in the cerebral cortex of the model mice, indicating that TSG has inhibitory effect on the over expression and aggregation of alpha -syn in the brain of alpha -syn transgenic mice. We found that the 15 month old a53t alpha -syn transgenic model group has the striatum, the cerebral cortex in the mice. The level of phosphorylation of alpha -syn at ser129 loci increases, and TSG can reduce the phosphorylation level of alpha -syn in the striatum and cerebral cortex of model mice (ser129 site), suggesting that TSG can improve posttranslational modification of alpha -syn, which may be one of the mechanisms of TSG inhibition of alpha -syn aggregation, and.5.tsg can increase the brain of 15 month old a53t alpha -syn transgenic mice. The expression of parkin protein, ATG5 and LC3 in the cortex and cerebellum, suggesting that TSG may enhance the degradation of alpha -syn by up regulation of the ubiquitin proteasome system (USP) and autophagy pathway, which may be another mechanism of inhibition of the over expression and aggregation of alpha -syn by TSG. In vitro, TSG can inhibit alpha -syn gene transfection into SH-SY5Y cells. Expression, reduce the phosphorylation level of alpha -syn at ser129 site and the phosphorylation level of PP2A in the cell model of rotenone compound alpha -syn gene transfection, suggesting that TSG may increase the activity of PP2A by down-regulation of PP2A phosphorylation, which may be one of the mechanisms of TSG inhibition of alpha -syn phosphorylation. Conclusion TSG can improve 15 month old A53T alpha -s. The exercise ability and memory function of YN transgenic mice, increase the number of dopaminergic neurons in substantia nigra, inhibit the activation of substantia nigra and striatum microglia, inhibit the over expression and aggregation of alpha -syn in the brain; its mechanism may involve the inhibition of phosphorylation of alpha -syn by down-regulation of PP2A phosphorylation, by up regulation of the ubiquitin proteasome system and autophagy. The results suggest that TSG may be beneficial to the prevention of synuclein related neurodegenerative diseases. The results suggest that -syn may play an important role in the prevention and treatment of neurodegenerative diseases associated with synuclein.

【學(xué)位授予單位】：首都醫(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)】：R741

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本文編號(hào)：1886118