本文關(guān)鍵詞： 電脈沖 胰島素 淀粉樣變性 熒光 TEM 圓二色譜 拉曼光譜　出處：《華東師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)結(jié)構(gòu)失穩(wěn)后會(huì)發(fā)生錯(cuò)誤折疊、集聚或纖維化,不僅喪失正常功能,有時(shí)甚至?xí)a(chǎn)生生物毒性。例如神經(jīng)退行性疾病及二型糖尿病都是因?yàn)榈鞍踪|(zhì)纖維化引起。纖維化了的蛋白質(zhì)在溶液中具有和淀粉類似的性質(zhì),因此蛋白質(zhì)的纖維化過(guò)程又被稱為淀粉樣變性,以產(chǎn)生β-折疊鏈狀的纖維絲為主要特征。電磁輻照是引起蛋白質(zhì)結(jié)構(gòu)失穩(wěn)的一個(gè)重要因素,經(jīng)電場(chǎng)輻照蛋白質(zhì)會(huì)發(fā)生去折疊和集聚,進(jìn)而改變自發(fā)折疊機(jī)制,因此經(jīng)受電場(chǎng)輻照的擾動(dòng)而結(jié)構(gòu)失穩(wěn)的蛋白質(zhì),纖維化過(guò)程會(huì)受到一定的影響。胰島素的分子結(jié)構(gòu)簡(jiǎn)單且具有非常明顯的成纖維趨向,被廣泛應(yīng)用于蛋白質(zhì)淀粉樣變性的相關(guān)研究。本文以體外胰島素溶液為研究對(duì)象,通過(guò)60℃恒溫加熱促進(jìn)胰島素在pH 2.0的HCl溶液中發(fā)生淀粉樣變性。利用硫磺素T(thioflavine T,ThT)染色的熒光光譜法從胰島素淀粉樣變性的宏觀動(dòng)力學(xué)過(guò)程方面、透射電子顯微鏡(transmission electron microscopy, TEM)從終產(chǎn)物成熟纖維絲的形貌變化方面、圓二色譜(circular dichroism, CD)法及拉曼光譜(Raman)法從原纖維絲二級(jí)結(jié)構(gòu)的角度,分別探究了50 Hz脈沖電場(chǎng)(pulsed intense electric fields, PEFs)不同輻照時(shí)間、33 Hz PEF不同持續(xù)時(shí)間及不同電場(chǎng)強(qiáng)度輻照對(duì)離體蛋白質(zhì)發(fā)生淀粉樣變性的纖維化機(jī)制的影響。各項(xiàng)研究指標(biāo)的實(shí)驗(yàn)結(jié)果均表明,50 Hz和33 Hz PEF輻照都能引起溶液中原始胰島素分子的去折疊和集聚,使得胰島素淀粉樣變性初期產(chǎn)生前體蛋白的成核期延長(zhǎng),原纖維絲聚集延長(zhǎng)過(guò)程(即中間產(chǎn)物的壽命)縮短,形成的成熟纖維絲長(zhǎng)度變短且整齊成簇不易糾纏。輻照時(shí)間增加、脈沖電場(chǎng)增強(qiáng),上述效應(yīng)也隨之增強(qiáng),這些結(jié)果一致說(shuō)明PEF輻照對(duì)胰島素的淀粉樣變性有抑制作用。但50 Hz和33 Hz PEF因脈沖波形不同,產(chǎn)生影響的機(jī)理不同：50 Hz PEF輻照過(guò)程有熱協(xié)同,而33 Hz PEF輻照的影響主要是電場(chǎng)作用的非熱效應(yīng)。延長(zhǎng)輻照時(shí)間與增加電場(chǎng)強(qiáng)度產(chǎn)生的抑制作用不盡相同：延長(zhǎng)輻照時(shí)間,發(fā)生聚集的胰島素分子比例增加但是聚合物尺寸變��；增加電場(chǎng)強(qiáng)度,胰島素分子則是由球狀去折疊為部分打開(kāi)的無(wú)規(guī)態(tài)。在胰島素淀粉樣變性的不同階段進(jìn)行輻照,抑制作用也不相同：輻照后胰島素的淀粉樣變性變得困難,成纖維動(dòng)力學(xué)過(guò)程縮短；而對(duì)已經(jīng)發(fā)生淀粉樣變性的胰島素原纖維輻照,其團(tuán)聚的成核中心解聚,且胰島素原纖維的成纖維活性降低。
[Abstract]:When the protein structure is unstable, misfolding, agglomeration or fibrosis will occur, and not only will it lose its normal function, Sometimes it can even be biotoxic. For example, neurodegenerative diseases and type 2 diabetes are caused by protein fibrosis, which has properties similar to starch in the solution. Therefore, the process of protein fibrosis is also called amyloidosis, which is characterized by the production of 尾 -folded chain-like filaments. Electromagnetic irradiation is an important factor causing the structural instability of proteins. Proteins irradiated by an electric field will be unfolded and concentrated, thus changing the mechanism of spontaneous folding. Therefore, proteins which are disturbed by electric field irradiation and whose structure is unstable, The molecular structure of insulin is simple and has a very obvious tendency of fibroblast, which is widely used in the study of protein amyloidosis. The isothermal heating at 60 鈩，

本文編號(hào)：1516856