本文選題：失眠　切入點：G蛋白偶聯(lián)受體　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：失眠(insomnia)是一種常見的睡眠障礙性疾病,在人群中患病率極高,不僅影響人們生活質(zhì)量,而且與憂郁、焦慮等疾病密切相關(guān)。進一步發(fā)展能夠引發(fā)多種代謝疾病,如:2型糖尿病。人體的睡眠由一系列受體調(diào)控,它們主要包括:GABAA受體、褪黑素受體(Melatonin受體)、五羥色胺受體(Serotonin受體)、食欲肽受體(Orexin受體)、組胺受體(Histamine受體)、腺苷受體(Adenosine受體)和多巴胺受體(Dopamine受體)。除GABAA受體外,其余的幾種受體都屬于G蛋白偶聯(lián)受體。市售治療失眠藥物主要針對GABAA受體來發(fā)揮作用,因為GABAA受體在神經(jīng)系統(tǒng)中彌散性分布,所以此類藥物彌散性地抑制中樞神經(jīng)系統(tǒng)并阻斷了腦干網(wǎng)狀結(jié)構(gòu)上行激活系統(tǒng)的傳導(dǎo)功能,從而使大腦皮質(zhì)細胞由興奮轉(zhuǎn)為抑制。但是,此類藥物有的會引起戒斷綜合癥以及具有白天殘留效應(yīng)等不良影響。目前治療失眠的新型藥物比如褪黑素(MT1/MT2)受體激動劑、五羥色胺(5HT2A)受體調(diào)節(jié)劑和食欲肽(OXR)受體拮抗劑都是特異性的作用于G蛋白偶聯(lián)受體(GPCR),不影響GABAA受體,在調(diào)節(jié)睡眠行為時副作用較少。本研究利用熒光共振能量轉(zhuǎn)移的技術(shù)(fluorescence resonance energy transfer,FRET)構(gòu)建以GPCR空間結(jié)構(gòu)變化的分子探針以及誘導(dǎo)表達受體的方法,可以鑒定化合物是否對GPCR有特異性作用,從而篩選出可能具有鎮(zhèn)靜安神作用的化合物來進行藥物開發(fā)。本課題選用五羥色胺5HT2A/1B受體、多巴胺D2/D3受體和食欲肽OX2受體三種與睡眠調(diào)節(jié)相關(guān)的G蛋白偶聯(lián)受體進行研究。首先,構(gòu)建多巴胺D2/D3受體表達載體和帶有熒光基團的5HT2A/1B受體分子探針載體,為構(gòu)建穩(wěn)定細胞株做基礎(chǔ)。然后,建立誘導(dǎo)表達多巴胺D2/D3受體和五羥色胺5HT2A/1B受體探針的穩(wěn)定細胞株,從而構(gòu)建出以多巴胺D2/D3受體和五羥色胺5HT2A/1B受體為預(yù)設(shè)靶點的藥物篩選平臺。最后,我們利用實驗室已有的誘導(dǎo)表達食欲肽OX2受體的穩(wěn)定細胞株對一系列化合物活性進行了篩選。研究結(jié)果表明,成功構(gòu)建了以多巴胺D2/D3受體和五羥色胺5HT2A/1B受體為預(yù)設(shè)靶點的藥物篩選平臺,為治療失眠藥物的開發(fā)提供了理論支持。另外,利用誘導(dǎo)表達食欲肽OX2受體的穩(wěn)定細胞株對化合物活性進行篩選,從一系列食欲肽受體拮抗性化合物(與中科院上海藥物所合作)中篩選出了多種可能具有鎮(zhèn)靜安神作用的有效成分,為靶向食欲肽受體的治療失眠藥物研究提供了數(shù)據(jù)支持。
[Abstract]:Insomnia omniais is a kind of common sleep disorder disease, which has a high prevalence rate in the crowd. It not only affects people's quality of life, but also is closely related to depression, anxiety and other diseases. Further development can lead to a variety of metabolic diseases. For example, in type 2 diabetes, sleep in the body is regulated by a series of receptors, which mainly include the receptor: GABAA. Melatonin receptor, serotonin receptor, orexin receptor, histamine receptor histamine receptor, adenosine receptor, dopamine receptor, and dopamine receptor, with the exception of GABAA receptor, melatonin receptor, serotonin receptor, orexin receptor, histamine receptor, adenosine receptor and dopamine receptor. The rest of the receptors are G-protein-coupled receptors. Marketed insomnia drugs mainly target GABAA receptors, because GABAA receptors are distributed diffusely in the nervous system. So this kind of drug diffusely inhibits the central nervous system and blocks the conduction function of the upstream activation system of the brain stem reticular structure, which changes the brain cortex cells from excitement to inhibition. Some of these drugs have adverse effects such as withdrawal syndrome and daytime residual effects. New drugs for insomnia, such as melatonin 1 / MT2) receptor agonists, Both serotonin 5HT2A) receptor modulators and orexin OXR receptor antagonists are specific to the G protein-coupled receptor (GPC) receptor, and do not affect the GABAA receptor. There are few side effects in regulating sleep behavior. In this study, fluorescence resonance energy transfer technique was used to construct molecular probes with spatial structure changes of GPCR and to induce expression of receptors. It is possible to identify whether the compounds have specific effects on GPCR and to screen out compounds that may have calming and soothing effects for drug development. This study selected serotonin 5HT2A / 1B receptors. Dopamine D _ 2 / D _ 3 receptor and appetite peptide OX2 receptor were used to study three G protein-coupled receptors associated with sleep regulation. Firstly, the dopamine D _ 2 / D _ 3 receptor expression vector and the 5HT _ 2A / 1B receptor molecular probe vector with fluorescence group were constructed. In order to construct a stable cell line, a stable cell line was established to induce the expression of dopamine D 2 / D 3 receptor and serotonin 5 HT 2 A / 1B receptor probe. So we build a drug screening platform with dopamine D2 / D3 receptor and serotonin 5HT2A / 1B receptor as the preset target. A series of compounds were screened using stable cell lines that induce orexin OX2 receptor expression in our laboratory. A drug screening platform with dopamine D _ 2 / D _ 3 receptor and serotonin _ 5HT _ 2A / 1B receptor as preset target was successfully constructed, which provides theoretical support for the development of drugs for treating insomnia. The activity of the compound was screened by the stable cell line which induced the expression of orexin OX2 receptor. From a series of orexin receptor antagonistic compounds (in cooperation with Shanghai Institute of Pharmacology, Chinese Academy of Sciences), a variety of active components with calming and tranquilizing effect were selected, which provided data support for the study on the treatment of insomnia by targeting appetite peptide receptor.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R740

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