ADTIQ的形成及其在糖尿病与帕金森病之间存在作用的研究

发布时间：2018-03-07 09:07

本文选题：1-乙酰-6　切入点：7-二羟基-1　出处：《北京理工大学》2014年博士论文　论文类型：学位论文

【摘要】：糖尿病(diabetes mellitus,DM)和帕金森病(Parkinson’s disease,PD)是两种世界性常见和高发的慢性疾病,严重威胁着人类的健康和生命。最近连续发表在糖尿病研究领域权威杂志《Diabetes Care》的3篇流行病学的调查结果显示:随着2型糖尿病的发病率的持续上升,患者患PD的几率也呈现上升的趋势,且2型糖尿病患者比正常人患PD的几率要高83%以上。近年来,有关糖尿病与中枢系统疾病,尤其是与PD之间的关系逐渐受到重视。在糖尿病患者中发现,病人的轴性体征、强直以及震颤均有不同程度的加重,提示着糖尿病与PD发生的相关性,糖尿病可能是诱导PD发生的一种危险因素。2010年,Morris等人研究发现2型糖尿病大鼠模型中黑质致密部(DSN)出现铁沉积,多巴胺(dopamine,DA)能神经元受损,及纹状体多巴胺分泌减少,推测有可能是因为高糖作用下,黑质致密部(DSN)铁沉积导致的氧化应激的增加,而多巴胺能神经元是对氧化应激敏感的神经元,更容易受到铁沉积毒素的作用,从而使2型糖尿病大鼠出现PD病理特征。尽管如此,有关糖尿病并发PD的具体机制还不清晰。我们课题组在PD病人脑中首次发现1-乙酰-6,7-二羟基-1,2,3,4-四氢异喹啉(1-acetyl-6,7-dihyroxy-1,2,3,4-Tetrahydro-isoquinoline,ADTIQ),其结构与1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)类似。由于ADTIQ是多巴胺与丙酮醛通过Pictet-Spengler(P-S)反应生成的产物,丙酮醛与葡萄糖代谢密切相关,因此我们推测ADTIQ有可能在糖尿病并发PD的过程中发挥关键作用。本课题围绕ADTIQ展开了物质合成,方法建立,含量检测,毒性实验等研究,探索糖尿病并发PD的致病机制。主要研究内容和研究结果如下:1.由于ADTIQ没有商业化的纯品,因此本课题首先完成了ADTIQ的化学合成与鉴定:采用Pictet-Spengler法,由多巴胺和丙酮醛一步合成ADTIQ,通过半制备高效液相色谱法制备出纯品,并利用红外、质谱、核磁等方法进行结构鉴定。结果表明:以多巴胺和丙酮醛(1:10)为底物,氮气保护在三氟乙酸的缓冲液中,37℃反应24小时后。半制备HPLC收集ADTIQ纯品,冷冻干燥后得到纯度大约82%的ADTIQ。结构鉴定结果表明我们成功得到ADTIQ。2.由于ADTIQ在生物体内的含量低,不易检测,因此本课题建立了ADTIQ的HPLC-MS/MS(multi-reaction monitoring,MRM))的高灵敏度检测方法,为ADTIQ的有效和准确检测提供了技术保障。3.本课题以SH-SY5Y细胞为模型,探索ADTIQ在细胞中的形成条件。结果显示:外源性丙酮醛与神经元内储存的神经递质多巴胺不易发生反应生成ADTIQ;葡萄糖代谢异常增加导致内源性丙酮醛含量的增加,并与神经元内的多巴胺反应是ADTIQ生成的前提。4.本课题对高糖诱导的SH-SY5Y细胞模型和2型糖尿病Sprague-Dawley大鼠模型进行研究,探索高糖与ADTIQ生成的关系。结果显示:体外和体内模型中,高糖引起的多巴胺神经元功能受损,导致多巴胺分泌减少;同时,我们检测到SH-SY5Y细胞内以及糖尿病SD大鼠纹状体ADTIQ和前体丙酮醛含量升高。说明ADTIQ以及前体丙酮醛的形成与神经元的损伤相关。5.本课题以SH-SY5Y细胞为多巴胺能神经元模型,研究ADTIQ对多巴胺能神经元的损伤机制,并通过对α-Synuclein WT,α-Synuclein A30P和α-Synuclein A53T,研究转基因PD小鼠模型中ADTIQ含量的检测,探索α-synuclein过表达的PD模型中ADTIQ与PD的相关性。结果显示:在ADTIQ的刺激条件下,多巴胺能神经元中促凋亡蛋白Bax激活,抗凋亡蛋白Bcl-2表达降低,导致细胞色素C的释放,引起casepase-3切割的增加,最终引发线粒体损伤,促进细胞凋亡。此外,过表达突变型的α-Synuclein的PD小鼠中ADTIQ的含量较正常组和过表达野生型α-Synuclein组显著增加(p0.05),提示ADTIQ可能在突变型的α-synuclein聚集导致的PD发病过程中发挥了重要作用。SSAO(semicarbazied-sensitive amine oxidases)是一类对氨基脲敏感的胺氧化酶,SSAO导致的管病变在糖尿病的发病中起着至关重要的作用。本课题成功地建立了人类脐动脉内皮细胞(human umbilical arterial endothelial cell,HUAEC)过表达SSAO的细胞模型,并与原核E.coli BL21模型,细胞系HEK模型以及人脐动脉动脉组织中表达的SSAO进行表达量,表达活性以及表达形式的比较,为今后SSAO在糖尿病以及PD的病因学研究提供一个基础的工具细胞模型。本研究发现ADTIQ的形成与糖尿病及PD之间的联系具有相关性,为以后研究糖尿病并发PD的机制提供一条新的思路。
[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.

【学位授予单位】：北京理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1;R742.5

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