本文選題：1-乙酰-6　切入點(diǎn)：7-二羥基-1　出處：《北京理工大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：糖尿病(diabetes mellitus,DM)和帕金森病(Parkinson’s disease,PD)是兩種世界性常見和高發(fā)的慢性疾病,嚴(yán)重威脅著人類的健康和生命。最近連續(xù)發(fā)表在糖尿病研究領(lǐng)域權(quán)威雜志《Diabetes Care》的3篇流行病學(xué)的調(diào)查結(jié)果顯示:隨著2型糖尿病的發(fā)病率的持續(xù)上升,患者患PD的幾率也呈現(xiàn)上升的趨勢,且2型糖尿病患者比正常人患PD的幾率要高83%以上。近年來,有關(guān)糖尿病與中樞系統(tǒng)疾病,尤其是與PD之間的關(guān)系逐漸受到重視。在糖尿病患者中發(fā)現(xiàn),病人的軸性體征、強(qiáng)直以及震顫均有不同程度的加重,提示著糖尿病與PD發(fā)生的相關(guān)性,糖尿病可能是誘導(dǎo)PD發(fā)生的一種危險(xiǎn)因素。2010年,Morris等人研究發(fā)現(xiàn)2型糖尿病大鼠模型中黑質(zhì)致密部(DSN)出現(xiàn)鐵沉積,多巴胺(dopamine,DA)能神經(jīng)元受損,及紋狀體多巴胺分泌減少,推測有可能是因?yàn)楦咛亲饔孟?黑質(zhì)致密部(DSN)鐵沉積導(dǎo)致的氧化應(yīng)激的增加,而多巴胺能神經(jīng)元是對氧化應(yīng)激敏感的神經(jīng)元,更容易受到鐵沉積毒素的作用,從而使2型糖尿病大鼠出現(xiàn)PD病理特征。盡管如此,有關(guān)糖尿病并發(fā)PD的具體機(jī)制還不清晰。我們課題組在PD病人腦中首次發(fā)現(xiàn)1-乙酰-6,7-二羥基-1,2,3,4-四氫異喹啉(1-acetyl-6,7-dihyroxy-1,2,3,4-Tetrahydro-isoquinoline,ADTIQ),其結(jié)構(gòu)與1-甲基-4-苯基-1,2,3,6-四氫吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)類似。由于ADTIQ是多巴胺與丙酮醛通過Pictet-Spengler(P-S)反應(yīng)生成的產(chǎn)物,丙酮醛與葡萄糖代謝密切相關(guān),因此我們推測ADTIQ有可能在糖尿病并發(fā)PD的過程中發(fā)揮關(guān)鍵作用。本課題圍繞ADTIQ展開了物質(zhì)合成,方法建立,含量檢測,毒性實(shí)驗(yàn)等研究,探索糖尿病并發(fā)PD的致病機(jī)制。主要研究內(nèi)容和研究結(jié)果如下:1.由于ADTIQ沒有商業(yè)化的純品,因此本課題首先完成了ADTIQ的化學(xué)合成與鑒定:采用Pictet-Spengler法,由多巴胺和丙酮醛一步合成ADTIQ,通過半制備高效液相色譜法制備出純品,并利用紅外、質(zhì)譜、核磁等方法進(jìn)行結(jié)構(gòu)鑒定。結(jié)果表明:以多巴胺和丙酮醛(1:10)為底物,氮?dú)獗Ｗo(hù)在三氟乙酸的緩沖液中,37℃反應(yīng)24小時(shí)后。半制備HPLC收集ADTIQ純品,冷凍干燥后得到純度大約82%的ADTIQ。結(jié)構(gòu)鑒定結(jié)果表明我們成功得到ADTIQ。2.由于ADTIQ在生物體內(nèi)的含量低,不易檢測,因此本課題建立了ADTIQ的HPLC-MS/MS(multi-reaction monitoring,MRM))的高靈敏度檢測方法,為ADTIQ的有效和準(zhǔn)確檢測提供了技術(shù)保障。3.本課題以SH-SY5Y細(xì)胞為模型,探索ADTIQ在細(xì)胞中的形成條件。結(jié)果顯示:外源性丙酮醛與神經(jīng)元內(nèi)儲(chǔ)存的神經(jīng)遞質(zhì)多巴胺不易發(fā)生反應(yīng)生成ADTIQ;葡萄糖代謝異常增加導(dǎo)致內(nèi)源性丙酮醛含量的增加,并與神經(jīng)元內(nèi)的多巴胺反應(yīng)是ADTIQ生成的前提。4.本課題對高糖誘導(dǎo)的SH-SY5Y細(xì)胞模型和2型糖尿病Sprague-Dawley大鼠模型進(jìn)行研究,探索高糖與ADTIQ生成的關(guān)系。結(jié)果顯示:體外和體內(nèi)模型中,高糖引起的多巴胺神經(jīng)元功能受損,導(dǎo)致多巴胺分泌減少;同時(shí),我們檢測到SH-SY5Y細(xì)胞內(nèi)以及糖尿病SD大鼠紋狀體ADTIQ和前體丙酮醛含量升高。說明ADTIQ以及前體丙酮醛的形成與神經(jīng)元的損傷相關(guān)。5.本課題以SH-SY5Y細(xì)胞為多巴胺能神經(jīng)元模型,研究ADTIQ對多巴胺能神經(jīng)元的損傷機(jī)制,并通過對α-Synuclein WT,α-Synuclein A30P和α-Synuclein A53T,研究轉(zhuǎn)基因PD小鼠模型中ADTIQ含量的檢測,探索α-synuclein過表達(dá)的PD模型中ADTIQ與PD的相關(guān)性。結(jié)果顯示:在ADTIQ的刺激條件下,多巴胺能神經(jīng)元中促凋亡蛋白Bax激活,抗凋亡蛋白Bcl-2表達(dá)降低,導(dǎo)致細(xì)胞色素C的釋放,引起casepase-3切割的增加,最終引發(fā)線粒體損傷,促進(jìn)細(xì)胞凋亡。此外,過表達(dá)突變型的α-Synuclein的PD小鼠中ADTIQ的含量較正常組和過表達(dá)野生型α-Synuclein組顯著增加(p0.05),提示ADTIQ可能在突變型的α-synuclein聚集導(dǎo)致的PD發(fā)病過程中發(fā)揮了重要作用。SSAO(semicarbazied-sensitive amine oxidases)是一類對氨基脲敏感的胺氧化酶,SSAO導(dǎo)致的管病變在糖尿病的發(fā)病中起著至關(guān)重要的作用。本課題成功地建立了人類臍動(dòng)脈內(nèi)皮細(xì)胞(human umbilical arterial endothelial cell,HUAEC)過表達(dá)SSAO的細(xì)胞模型,并與原核E.coli BL21模型,細(xì)胞系HEK模型以及人臍動(dòng)脈動(dòng)脈組織中表達(dá)的SSAO進(jìn)行表達(dá)量,表達(dá)活性以及表達(dá)形式的比較,為今后SSAO在糖尿病以及PD的病因?qū)W研究提供一個(gè)基礎(chǔ)的工具細(xì)胞模型。本研究發(fā)現(xiàn)ADTIQ的形成與糖尿病及PD之間的聯(lián)系具有相關(guān)性,為以后研究糖尿病并發(fā)PD的機(jī)制提供一條新的思路。
[Abstract]:Diabetes (diabetes mellitus, DM) and Parkinson disease (Parkinson s disease, PD) is a chronic disease of two world common and high, a serious threat to human health and life. The recent publication in Diabetes Research Journal of 3 epidemiological survey results show that: with the continuous the rising incidence of type 2 diabetes mellitus, probability of patients suffering from PD also showed a rising trend, and the risk of type 2 diabetic patients than normal with PD higher than 83%. In recent years, the diabetes and diseases of the central nervous system, especially the relationship between PD and has attracted much attention. Found in diabetic patients. The shaft of the tonic and signs of the patients, aggravate tremor in varying degrees, suggesting a correlation between diabetes and the incidence of PD, PD induced diabetes may be a risk factor for.2010 years, Morris found that 2 The model of diabetic rats in the substantia nigra (DSN) iron deposition, dopamine (dopamine, DA) can be damaged neurons and striatal dopamine secretion decreased, possibly because the effect of high glucose, substantia nigra (DSN) increased oxidative stress leads to iron deposition, and dopaminergic neurons of neurons are sensitive to oxidative stress, more susceptible to iron deposition in the role of the toxin, so that the type 2 diabetic rats PD pathological characteristics. However, the specific mechanism of diabetes mellitus complicated with PD is still not clear. Our research group for the first time that 1- acetyl -6,7- two hydroxy -1,2,3,4- four isoquinoline in the brain of PD patients (1-acetyl-6,7-dihyroxy-1,2,3,4-Tetrahydro-isoquinoline, in ADTIQ), and the structure of 1- methyl -4- phenyl -1,2,3,6- tetrahydropyridine four (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP). ADTIQ is due to dopamine and methylglyoxal The Pictet-Spengler (P-S) reaction products, closely related to glucose metabolism aldehyde and acetone, so we speculate that ADTIQ may play a key role in the process of diabetes complicated with PD. The subject matter synthesis, around the establishment of ADTIQ, the content of detection, experimental study on the toxicity and the pathogenesis of diabetes mellitus complicated with PD. The main exploration the research contents and results are as follows: 1. because the ADTIQ has no commercial pure product, so this paper completed the chemical synthesis and identification of ADTIQ: the Pictet-Spengler method by one step synthesis of ADTIQ dopamine and acetone aldehyde chromatography were prepared in pure form by semi preparative HPLC, mass spectrometry, and infrared. The structure identification of NMR method. The results show that using dopamine and methylglyoxal (1:10) as substrate, buffer liquid nitrogen protection in three fluorine acetic acid, 24 hours after the 37 C reaction. Semi preparative HPLC In pure ADTIQ, freeze drying, the purity of about 82% ADTIQ. identification results showed that we successfully obtained ADTIQ.2. as the content of ADTIQ in organism is low, not easy to detect, so this research established the ADTIQ HPLC-MS/MS (multi-reaction) monitoring, MRM) high sensitivity detection method, ADTIQ is effective and accurate detection technical support for.3. this topic in SH-SY5Y cells as a model, to explore the formation conditions of ADTIQ in the cells. The results showed that the neurotransmitter dopamine neurons and exogenous methylglyoxal storage is not easy to react to ADTIQ; increased glucose metabolism leads to increased endogenous acetone aldehyde content, and with the dopamine response in neurons is a prerequisite.4. ADTIQ generated the study on the model of SH-SY5Y cell model induced by high glucose and type 2 diabetes Sprague-Dawley rats of high glucose and ADTI The relationship between Q formation. The results showed that in vitro and in vivo models, the function of dopamine neurons induced by high glucose impaired, leading to dopamine secretion decreased; at the same time, we detected SH-SY5Y and ADTIQ cells in diabetic SD rat striatum and precursors of methylglyoxal content increased. The results showed that ADTIQ and neuronal precursors for the formation of methylglyoxal injury. In this study.5. using SH-SY5Y cells as dopaminergic neuron model to study the mechanism of ADTIQ damage to dopaminergic neurons, and through the -Synuclein WT alpha, alpha -Synuclein alpha A30P and -Synuclein A53T, PD ADTIQ in detection of transgenic mice, and explore the correlation between -synuclein ADTIQ and PD PD alpha expression in the model. The results showed that in ADTIQ conditions of stimulation of dopaminergic neurons in the pro apoptotic protein Bax activation, anti apoptosis protein Bcl-2 expression decreased, resulting in the release of cytochrome C, Due to the increase of casepase-3 cutting, eventually lead to mitochondrial damage, promote cell apoptosis. In addition, the content of ADTIQ over expression of alpha -Synuclein mutant PD mice than in normal group and overexpression of wild-type alpha -Synuclein group increased significantly (P0.05),.SSAO played an important role that alpha -synuclein ADTIQ in mutant aggregation cause the pathogenesis of PD (semicarbazied-sensitive amine oxidases) is a kind of semicarbazide sensitive amine oxidase, tube disease caused by SSAO plays an important role in the onset of diabetes. This study successfully established human umbilical artery endothelial cells (human umbilical arterial endothelial cell, HUAEC) over expression model of SSAO. And with the prokaryotic expression cell line E.coli BL21 model, HEK model and human umbilical artery tissues SSAO expression, expression activity and expression This study will provide a basic tool cell model for the future research of SSAO in the etiology of diabetes and PD. This study found that the formation of ADTIQ is correlated with the relationship between diabetes and PD, and provides a new way for studying the mechanism of diabetes complicated with PD in the future.

【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】：2014
【分類號】：R587.1;R742.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 毛健;徐妍;鄧玉林;林凡凱;謝冰潔;王睿;;急性酒精中毒的鼠腦內(nèi)乙醛及其單胺神經(jīng)遞質(zhì)縮合物的測定[J];分析化學(xué);2010年12期

2 宋德偉;杜合泉;王琳;胡高飛;鄧玉林;;HPLC-ESI-MS對糖尿病大鼠腦紋狀體與海馬區(qū)兒茶酚異喹啉類化合物的檢測[J];化學(xué)通報(bào);2010年11期

3 宋德偉;胡高飛;周筠;王洪彬;王琳;朱勇;鄧玉林;;糖尿病大鼠紋狀體、海馬區(qū)帕金森病相關(guān)蛋白的蛋白質(zhì)組學(xué)初步研究[J];化學(xué)通報(bào);2008年06期

4 王冉;慶宏;劉曉茜;鄭曉琳;鄧玉林;;鐵離子促進(jìn)過量多巴胺引起SH-SY5Y細(xì)胞兒茶酚異喹啉物質(zhì)生成和氧化損傷(英文)[J];Neuroscience Bulletin;2008年03期

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