本文選題：胰島淀粉樣多肽 + 淀粉樣纖維��； 參考：《鄭州大學(xué)》2017年博士論文

【摘要】：構(gòu)象病是由于蛋白質(zhì)錯(cuò)誤折疊引起的一種疾病,其典型特征為具有正常功能的蛋白質(zhì)構(gòu)型發(fā)生非正常變化,引起錯(cuò)誤折疊,導(dǎo)致蛋白質(zhì)從正常形態(tài)逐漸變?yōu)楦缓罅喀抡郫B結(jié)構(gòu)的淀粉樣斑塊,沉積在組織器官中,從而使細(xì)胞功能衰退直至凋亡。2型糖尿病作為一種常見(jiàn)的構(gòu)象病,由于它患病人數(shù)逐年增加,已經(jīng)成為一種嚴(yán)重危害人類(lèi)生活質(zhì)量和身體健康的流行性疾病。胰島淀粉樣多肽的纖維化沉積被認(rèn)為是2型糖尿病的主要病理特征之一。雖然口服降糖藥以及直接注射胰島素均可用于治療2型糖尿病,但是此種干預(yù)性治療只能初步改善和維持正常血糖值,在治療后期,隨著并發(fā)癥逐漸發(fā)生,此種治療方法具有一定局限性。胰島淀粉樣多肽聚集形成的淀粉樣纖維能夠破壞胰島β細(xì)胞膜,導(dǎo)致β細(xì)胞逐漸凋亡,加劇了2型糖尿病的病癥。抑制胰島淀粉樣多肽的聚集能夠減緩2型糖尿病的病發(fā),因此研發(fā)抑制胰島淀粉樣多肽聚集抑制劑為2型糖尿病的治療提供了一種新方法。人胰島淀粉樣多肽(hIAPP)的不同片段具有不同性質(zhì),阻斷hIAPP自身π-π堆積作用,可以有效減弱hIAPP成淀粉樣纖維的能力。因此本論文針對(duì)人胰島淀粉樣多肽自聚集行為進(jìn)行了研究并設(shè)計(jì)合成了多種多肽類(lèi)抑制劑。首先,本研究以牛血清白蛋白(BSA)為模版蛋白,運(yùn)用微量熱泳動(dòng)技術(shù)(MST)建立了一種能夠研究蛋白聚集行為的方法,考察了人胰島淀粉樣多肽片段_(11-20)(hIAPP_(11-20))在不同溶液中聚集行為,最終以hIAPP_(11-20)為目標(biāo)物,設(shè)計(jì)并合成了一系列具有不同構(gòu)型的多肽,考察了它們對(duì)hIAPP_(11-20)淀粉樣纖維形成的抑制效果,探討了影響hIAPP_(11-20)淀粉樣纖維形成的主要因素。具體研究?jī)?nèi)容如下:1.運(yùn)用微量熱泳動(dòng)技術(shù)建立研究蛋白聚集行為的方法在藥物研發(fā)過(guò)程中,探究蛋白與其目標(biāo)分子的結(jié)合行為為藥物分子設(shè)計(jì)提供了大量的基本信息。本實(shí)驗(yàn)以牛血清白蛋白(BSA)為模板蛋白,分別運(yùn)用微量熱泳動(dòng)技術(shù)(MST)和普通熒光猝滅方法,考察了BSA與一種常見(jiàn)小分子異硫氰酸鈉(FITC)之間的相互作用。MST實(shí)驗(yàn)表明,在短時(shí)間內(nèi),BSA與FITC發(fā)生了兩種形式的相互作用。在MST競(jìng)爭(zhēng)實(shí)驗(yàn)中,以華法林和布洛芬分別為BSA不同位點(diǎn)的特異性結(jié)合標(biāo)記物,結(jié)果表明,FITC主要與BSA的位點(diǎn)II位結(jié)合,但也與位點(diǎn)I位有少量結(jié)合,同時(shí)也證明了FITC并不以標(biāo)記的形式與BSA發(fā)生相互作用,而是與BSA聚集體發(fā)生結(jié)合,從而表現(xiàn)出2種結(jié)合方式。MST實(shí)驗(yàn)所得的結(jié)果與熒光實(shí)驗(yàn)結(jié)果相一致,進(jìn)一步表明MST在研究生物分子間相互作用中的可靠性。因此,MST不僅可以為生物分子間相互作用提供大量有用的信息,也可以研究蛋白聚集行為,獲得其它方法不能得到的信息。2.hIAPP_(11-20)在不同溶液中的聚集行為研究hIAPP_(11-20)能夠在體外形成與全長(zhǎng)類(lèi)似的富含β折疊的淀粉樣纖維,因此選擇hIAPP_(11-20)作為探究淀粉樣多肽聚集行為的目標(biāo)物。本實(shí)驗(yàn)運(yùn)用MST技術(shù)和傳統(tǒng)的Th T熒光法,考察了hIAPP_(11-20)在不同溶液(Tris-HCl,HEPES,PBS和超純水)中的淀粉樣纖維的形成過(guò)程。實(shí)驗(yàn)結(jié)果表明,hIAPP_(11-20)在不同溶液中表現(xiàn)出不同的聚集行為,hIAPP_(11-20)不僅與自身發(fā)生相互作用,也可能與溶質(zhì)分子發(fā)生結(jié)合,說(shuō)明緩沖溶液不僅只是起到維持溶液環(huán)境穩(wěn)定的作用,還有可能參與目標(biāo)分子的相互作用。hIAPP_(11-20)與Tris的親和力遠(yuǎn)大于hIAPP_(11-20)與其它溶質(zhì)分子的親和力,說(shuō)明hIAPP_(11-20)在Tris-HCl緩沖溶液中自聚能力要遠(yuǎn)小于hIAPP_(11-20)在其它溶液中的自聚能力。在HEPES和超純水中,hIAPP_(11-20)表現(xiàn)出相似的自聚集行為。PBS緩沖溶液中帶負(fù)電的磷酸根離子可能會(huì)“中和”一部分hIAPP_(11-20)自身的正電,從而導(dǎo)致hIAPP_(11-20)在PBS中快速形成淀粉樣纖維聚集。因此,在研究hIAPP_(11-20)淀粉樣纖維化過(guò)程時(shí),緩沖溶液的影響需考慮在內(nèi)。同時(shí),MST技術(shù)也為研究多肽或蛋白的早期聚集行為提供了一種簡(jiǎn)單有效的方法。3.新型阻斷肽抑制hIAPP_(11-20)聚集效果的研究hIAPP錯(cuò)誤折疊所產(chǎn)生的淀粉樣纖維是2型糖尿病的一種重要病理特征。本實(shí)驗(yàn)設(shè)計(jì)并合成了17條具有不同長(zhǎng)度,構(gòu)型以及組成的多肽類(lèi)抑制劑,以hIAPP_(11-20)為模板,考察了它們抑制hIAPP_(11-20)淀粉樣纖維形成的效果。通過(guò)MST實(shí)驗(yàn)以及Th T熒光實(shí)驗(yàn)深入探究了這些多肽抑制hIAPP_(11-20)淀粉樣纖維形成的機(jī)理,并用TEM表征了hIAPP_(11-20)最終形成淀粉樣纖維的形貌。結(jié)果表明短肽AT、SA、RF、KS、KT和KN,以及環(huán)肽cyclic-KS、cyclic-KT和cyclic-KN能夠有效抑制hIAPP_(11-20)淀粉樣纖維的形成。其中,芳香型氨基酸的種類(lèi),多肽氨基酸的特定組成以及多肽的構(gòu)型在抑制淀粉樣纖維形成中起重要作用。MST實(shí)驗(yàn)量化了hIAPP_(11-20)與多肽之間的親和力,表明具有較高親和力的多肽在抑制聚集中表現(xiàn)出良好的抑制效果,該結(jié)果與Th T熒光實(shí)驗(yàn)以及分子對(duì)接結(jié)果相一致。因此,MST是一種簡(jiǎn)單快速的方法,可用于初步篩選聚集抑制劑。本研究也為以后設(shè)計(jì)多肽抑制劑提供了一些實(shí)驗(yàn)基礎(chǔ),同時(shí)也為探究其他蛋白構(gòu)象病的抑制劑擴(kuò)展了思路。4.脂肽抑制hIAPP_(11-20)聚集效果的研究根據(jù)有些脂肽能夠保護(hù)細(xì)胞免受淀粉樣纖維破環(huán),而且脂肽與報(bào)道的抑制劑均含有較多的疏水基團(tuán)等特點(diǎn),本實(shí)驗(yàn)設(shè)計(jì)了一系列新型脂肽,以hIAPP_(11-20)為探究淀粉樣多肽聚集的目標(biāo)物,考察了這些脂肽抑制hIAPP_(11-20)淀粉樣纖維形成的效果。我們通過(guò)MST、Th T熒光實(shí)驗(yàn)深入討論了這些脂肽抑制聚集的機(jī)理,同時(shí)利用TEM表征了hIAPP_(11-20)最終形成淀粉樣纖維的形貌及hIAPP_(11-20)與脂肽共同孵育后的hIAPP_(11-20)聚集形貌。結(jié)果表明,隨著脂肽鏈長(zhǎng)的逐漸增加,其抑制hIAPP_(11-20)聚集的效果逐漸增強(qiáng)。另外,含正電氨基酸的數(shù)量也在抑制hIAPP_(11-20)淀粉樣纖維形成中起到一定作用。因此,本實(shí)驗(yàn)拓寬了hIAPP淀粉樣纖維化抑制劑的研究范圍。本論文通過(guò)運(yùn)用MST建立了一種能夠研究蛋白聚集行為的方法,并采用該方法聯(lián)合使用其他技術(shù)考察了hIAPP_(11-20)在不同溶液中的自聚集行為,探討了不同類(lèi)型抑制劑的抑制hIAPP_(11-20)聚集的效果,總結(jié)出影響hIAPP_(11-20)淀粉樣纖維化的因素,
[Abstract]:Conformational disease is a disease caused by the wrong folding of proteins, typically characterized by abnormal changes in the structure of proteins with normal functions, causing false folds, causing proteins to gradually change from normal form to amyloid plaques rich in beta folded structures and accumulate in tissues and organs so that cell function decline straight. As a common conformation disease, apoptotic type.2 diabetes has become a popular disease that seriously endangers the quality of human life and health. The fibrotic deposition of islet amyloid peptide is considered to be one of the main pathological features of type 2 diabetes. Insulin can be used in the treatment of type 2 diabetes, but this intervention therapy can only improve and maintain normal blood glucose. In the later period of treatment, with the gradual occurrence of complications, this method has some limitations. The amyloid Fibrinosis formed by amyloid polypeptide of islet amyloid peptide can destroy the islet beta cell membrane and lead to beta cells gradually. Apoptosis, exacerbates the disease of type 2 diabetes. Inhibition of the aggregation of islet amyloid peptide can slow the onset of type 2 diabetes. Therefore, the development of the inhibition of islet amyloid peptide aggregation inhibitors provides a new method for the treatment of type 2 diabetes. The different fragments of the human islet amyloid polypeptide (hIAPP) have different properties and block the hIAPP itself - pion. Pion accumulation can effectively weaken the ability of hIAPP to form amyloid fibers. Therefore, this paper studies the self aggregation behavior of human islet amyloid peptide and designs a variety of polypeptide inhibitors. First, a kind of MST was established by using BSA as a template and a micro thermo swimming technique (MST). In the method of protein aggregation behavior, the aggregation behavior of human islet amyloid polypeptide fragment (11-20) (11-20) (hIAPP_ (11-20)) was investigated in different solutions. A series of polypeptides with different configurations were designed and synthesized with hIAPP_ (11-20) as the target, and their inhibition effect on the formation of hIAPP_ (11-20) amyloid fibers was investigated and the influence of hI was discussed. The main factors for the formation of APP_ (11-20) amyloid fiber are as follows: 1. the method of using microcalorimetry to establish the behavior of protein aggregation in the process of drug development, the combination of protein and its target molecules provides a large amount of basic information for the design of drug molecules. This experiment is based on bovine serum albumin (BSA). For template protein, the interaction between BSA and a common sodium isothiocyanate (FITC) was investigated by microcalorimetry (MST) and common fluorescence quenching. The interaction between BSA and FITC in a short time showed that there were two forms of interaction between BSA and FITC. In the MST competition experiment, Hua Falin and Bloven were respectively BSA The specific binding markers of different loci show that FITC is mainly associated with the II site of the site of BSA, but also has a small combination with the site I position. It also shows that FITC does not interact with BSA in the form of labeling, but is a combination with BSA aggregates, thus showing the results obtained from the 2 binding modes.MST experiment and fluorescence reality. The results are consistent, which further indicates the reliability of MST in the study of intermolecular interaction. Therefore, MST can not only provide a large number of useful information for intermolecular interaction, but also study the aggregation behavior of protein, and obtain the aggregation behavior of.2.hIAPP_ (11-20) in different solutions (hI), which are not obtained by other methods. APP_ (11-20) can form amyloid like fiber rich in beta fold in vitro, so hIAPP_ (11-20) is selected as a target to explore the aggregation behavior of amyloid polypeptide. The experiment used MST technology and traditional Th T fluorescence method to investigate the starch like fiber of hIAPP_ (11-20) in different solutions (Tris-HCl, HEPES, PBS, and ultra pure water). The experimental results show that hIAPP_ (11-20) shows different aggregation behavior in different solutions. HIAPP_ (11-20) not only interact with itself, but also combine with solute molecules, indicating that the buffer solution not only plays a role in maintaining the stability of the solution, but may also be involved in the interaction of the target molecules. The affinity between.HIAPP_ (11-20) and Tris is far greater than the affinity of hIAPP_ (11-20) to other solute molecules, indicating that the self polymerization of hIAPP_ (11-20) in Tris-HCl buffer solution is much smaller than that of hIAPP_ (11-20) in other solutions. In HEPES and super pure water, hIAPP_ (11-20) shows a similar self aggregation behavior in.PBS buffer solution. Negatively charged phosphate ions may "neutralize" a portion of hIAPP_ (11-20) itself, leading to the rapid formation of amyloid fibrils in hIAPP_ (11-20) in PBS. Therefore, the impact of the buffer solution needs to be taken into account in the study of hIAPP_ (11-20) amyloid fibrosis. At the same time, MST technology is also the early study of the peptide or protein. Aggregation behavior provides a simple and effective method of inhibition of hIAPP_ (11-20) aggregation effect of.3. new blocking peptide. The amyloid fiber produced by hIAPP error folding is an important pathological feature of type 2 diabetes. This experiment designed and synthesized 17 polypeptide inhibitors with different length, structure and composition, with hIAPP_ (11-2). 0) the effect of their inhibition on the formation of hIAPP_ (11-20) amyloid fibers was investigated for the template. The mechanism of inhibiting the formation of hIAPP_ (11-20) amyloid fibers by these peptides was investigated by MST and Th T fluorescence. The morphology of hIAPP_ (11-20) in the final formation of starch like fibers was characterized by TEM. The results showed that the short peptide AT, SA, RF, KS, KT and minerals were used. And cyclic peptide cyclic-KS, cyclic-KT and cyclic-KN can effectively inhibit the formation of hIAPP_ (11-20) amyloid fibers. Among them, the species of aromatic amino acids, the specific composition of polypeptide amino acids and the configuration of peptides play an important role in inhibiting the formation of amyloid fibers..MST experiments have quantified the affinity between hIAPP_ (11-20) and polypeptide. Polypeptides with high affinity exhibit good inhibitory effects in inhibition of aggregation. This result is consistent with the results of Th T fluorescence experiments and molecular docking. Therefore, MST is a simple and rapid method for preliminary screening of aggregation inhibitors. This study also provides some experimental basis for the future design of polypeptides. In order to explore other inhibitors of protein conformation disease, a study on the inhibition of.4. lipopeptides to inhibit the aggregation of hIAPP_ (11-20) is based on some lipopeptides that protect cells from amyloid fibrils, and both lipopeptides and reported inhibitors contain many hydrophobic groups. A series of new lipopeptides were designed in this experiment, with hIAPP_ (11-20). In order to explore the target of amyloid polypeptide aggregation, the effects of these lipopeptides on the formation of hIAPP_ (11-20) amyloid fibers were investigated. The mechanism of inhibiting aggregation of these lipopeptides was discussed through MST and Th T fluorescence experiments. At the same time, the morphology of hIAPP_ (11-20) amyloid fibers was characterized by TEM and hIAPP_ (11-20) was shared with lipopeptides. The aggregation morphology of hIAPP_ (11-20) after incubation showed that with the gradual increase of the length of the lipoprotein chain, the effect of its inhibition of hIAPP_ (11-20) aggregation increased gradually. In addition, the number of positive amino acids also played a role in inhibiting the formation of hIAPP_ (11-20) amyloid fibers. Therefore, this experiment broadened the hIAPP amyloid fibrosis inhibitor. In this paper, a method to study the aggregation behavior of protein was established by using MST, and the self aggregation behavior of hIAPP_ (11-20) in different solutions was investigated by using the method combined with other techniques. The effect of different inhibitors on the inhibition of hIAPP_ (11-20) aggregation was discussed, and the effect of hIAPP_ (11-20) starch samples was summarized. Factors of fibrosis,
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ460.1

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