本文關(guān)鍵詞：均一透明質(zhì)酸的酶法合成策略及生物學(xué)功能評價　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 透明質(zhì)酸 聚合度均一 酶法合成 嵌合型糖胺聚糖

【摘要】：糖胺聚糖(glycosaminoglycans,GAGs)是哺乳動物中普遍存在的天然雜多糖,結(jié)構(gòu)上由二糖重復(fù)單元連接而成,以蛋白聚糖或游離形式存在。糖胺聚糖的重要生理功能及其與重大疾病的密切關(guān)系近年來受到國內(nèi)外科學(xué)家的廣泛關(guān)注。透明質(zhì)酸(hyaluronan,HA)是結(jié)構(gòu)最簡單的糖胺聚糖,由二糖重復(fù)單位[-D-glucuronic acid-β 1,3-N-acetyl-D-glucosamine-β 1,4-]連接而成,不同于其他糖胺聚糖,透明質(zhì)酸沒有硫酸化修飾且不與蛋白共價結(jié)合,以游離形式存在。HA廣泛存在于動物組織及某些微生物中,由于其良好的保水性、粘彈性和生物相容性,已經(jīng)廣泛應(yīng)用于保健品、化妝品及醫(yī)藥等領(lǐng)域。近年來研究發(fā)現(xiàn),HA除了作為細(xì)胞外基質(zhì)的結(jié)構(gòu)組成外,還參與了細(xì)胞識別、信號轉(zhuǎn)導(dǎo)等重要生理過程,在腫瘤的發(fā)生、發(fā)展及炎癥反應(yīng)過程中發(fā)揮了多重生物效應(yīng)。代謝異常是腫瘤的重要特征,腫瘤微環(huán)境中,HA合成和降解的動態(tài)平衡發(fā)生異常,糖鏈長度變化是一個重要體現(xiàn),也是腫瘤惡性程度的評價指標(biāo)之一。腫瘤細(xì)胞表面存在大量HA特異性受體和結(jié)合蛋白,HA通過與受體或結(jié)合蛋白的相互作用,激活細(xì)胞內(nèi)多條信號通路,參與腫瘤發(fā)生發(fā)展和炎癥反應(yīng)等生物效應(yīng)過程。尤為值得關(guān)注的是,有別于其它生物效應(yīng)分子的合成后修飾過程,HA的生物活性受其糖鏈長度的調(diào)控,不同糖鏈長短的HA表現(xiàn)出截然不同的生物效應(yīng)。受生物體內(nèi)多糖合成過程的影響,商品化HA具有高度不均一性(分子量分布范圍較寬且呈彌散性分布),嚴(yán)重阻礙了HA生物學(xué)功能的正確表述。目前,商品化HA的生產(chǎn)方法主要有兩種。一是動物組織提取,這種技術(shù)產(chǎn)量較低,且存在動物原料受限和動物病毒殘存問題。二是微生物發(fā)酵,用這種方法生產(chǎn)HA成本低、規(guī)模大且不受原料的限制,但存在細(xì)菌內(nèi)毒素、核酸、蛋白質(zhì)及重金屬污染的風(fēng)險。上述方法在商品化HA的制備方面得到了較大發(fā)展,但在均一化HA的精細(xì)化制備方面還極具挑戰(zhàn)性。本論文圍繞均一 HA的制備這一科學(xué)問題,對巴斯德氏菌來源的透明質(zhì)酸合酶(PmHAS)的催化反應(yīng)機理進行了深入研究,采用逐步酶法合成HA寡糖片段和一步法同步化合成均一 HA,發(fā)展了均一 HA的兩步偶聯(lián)酶法合成策略。論文第二章節(jié)通過深入研究PmHAS的催化反應(yīng)機理,發(fā)現(xiàn)PmHAS催化新生HA糖鏈合成過程中存在起始限速步驟,造成了產(chǎn)物的高度不均一性。通過對PmHAS的底物選擇性分析,發(fā)現(xiàn)透明質(zhì)酸三糖是PmHAS催化合成均一 HA所必需的最小受體底物;進一步研究發(fā)現(xiàn),催化反應(yīng)體系中受體與供體的摩爾濃度比例是影響HA糖鏈長度的關(guān)鍵因素,通過對反應(yīng)體系中受體/供體摩爾濃度進行化學(xué)計量控制,實現(xiàn)了均一HA的糖鏈長度可控酶法合成。隨后進一步拓展了均一HA酶法合成策略在化學(xué)結(jié)構(gòu)明確、HA糖鏈長度可控的HA綴合物制備領(lǐng)域的應(yīng)用。制備了生物素標(biāo)記的透明質(zhì)酸(HA-biotin conjugates)以及透明質(zhì)酸-藥物綴合物(HA-oroxylin conjugates)。HA-biotin conjugates 能夠作為分子探針,對外源 HA的細(xì)胞內(nèi)代謝過程進行可視化檢測;HA-oroxylin conjugates作為模式藥物,為HA修飾抗腫瘤靶向藥物的研發(fā)奠定了基礎(chǔ)。論文第三章開展了透明質(zhì)酸-硫酸軟骨素(HA-CS)嵌合型糖胺聚糖的合成。由于HA與CS結(jié)構(gòu)的相似性及PmHAS靈活的底物適應(yīng)性,通過逐步反應(yīng)的方法合成了 CS2HA2-N3、HA2CS2HA2-N3、CS2HA4-N3 和 CS4HA4-N3 四種 HA-CS嵌合型糖胺聚糖,用于研究微觀結(jié)構(gòu)改變對HA生物功能的影響。該方法也可應(yīng)用于其他不同嵌合型糖胺聚糖的合成中,為嵌合型糖胺聚糖的生物功能研究奠定了基礎(chǔ)。論文第四章節(jié)中,結(jié)合本實驗室在糖核苷酸酶法合成方面的工作基礎(chǔ),建立了一種以單糖為起始的多酶偶聯(lián)的HA合成策略。以廉價單糖GlcNAc和GlcA為起始底物,將糖核苷酸的酶法合成與透明質(zhì)酸的聚合反應(yīng)相偶聯(lián),一鍋多酶催化高效合成HA。并利用此方法成功合成了 HA衍生物,用于交聯(lián)HA的制備和應(yīng)用研究,具有十分廣闊的應(yīng)用前景。
[Abstract]:Glycosaminoglycans (glycosaminoglycans, GAGs) are ubiquitous natural Heteropoly saccharides in mammals. They are made up of two sugar repeating units, and exist in proteoglycan or free form. The important physiological functions of glycosaminoglycan and its close relationship with major diseases have been widely concerned by scientists at home and abroad in recent years. Hyaluronic acid (hyaluronan, HA) is the most simple glycosaminoglycan structure, composed of two repeat units [-D-glucuronic acid- beta 1,3-N-acetyl-D-glucosamine- beta 1,4-] connection, different from other glycosaminoglycans, hyaluronic acid and no sulfated modification does not bind covalent protein, is present in a free form. HA is widely distributed in animal tissues and some microorganisms. Because of its good water retention, viscoelasticity and biocompatibility, it has been widely applied in health products, cosmetics and medicine. In recent years, studies have found that HA, as a structural component of extracellular matrix, is also involved in cell recognition, signal transduction and other important physiological processes. It plays multiple biological effects in the process of tumor occurrence, development and inflammatory response. Metabolic abnormality is an important feature of cancer. In the microenvironment of cancer, the dynamic balance of HA synthesis and degradation is abnormal. The change of sugar chain length is an important manifestation, and it is also an evaluation index of malignancy of tumor. A large number of HA specific receptors and binding proteins exist on the surface of tumor cells. HA activates many signaling pathways in cells through interaction with receptors or binding proteins, and participates in biological processes such as oncogenesis, development and inflammatory response. What is particularly noteworthy is that it is different from other biological effect molecules in the process of post modification. The biological activity of HA is regulated by the length of its sugar chain. HA with different sugar chain length shows different biological effects. Influenced by the process of polysaccharide synthesis, commercialized HA is highly heterogeneous, with a wide distribution of molecular weight and diffuse distribution, which seriously hampers the correct expression of HA's biological functions. At present, there are two main methods of production of commercialized HA. The first is the extraction of animal tissue, which is low in production, and there is a problem of animal raw material limitation and animal virus surviving. The two is microbial fermentation. In this way, the production of HA is low cost, large scale and not limited by raw materials, but it has the risk of bacterial endotoxin, nucleic acid, protein and heavy metal pollution. The above method has been greatly developed in the preparation of commercial HA, but it is still very challenging in the fine preparation of homogenized HA. This paper focuses on the preparation of homogeneous HA this scientific problem, hyaluronic acid synthase on Pasteur's source of bacteria (PmHAS) catalytic reaction mechanism were studied, using stepwise enzymatic synthesis of HA oligosaccharides and one-step synchronous combined Cheng Junyi HA, the two step development strategy coupled enzymatic synthesis of uniform HA. In the second chapter, through in-depth study of the catalytic mechanism of PmHAS, it is found that PmHAS catalyzes the initial rate limiting steps in the process of HA synthesis, resulting in highly heterogeneous products. Based on the PmHAS substrate selectivity analysis found that hyaluronic acid three sugar is required for PmHAS catalytic synthesis of homogeneous HA minimal receptor substrate; further study found that the ratio of molar concentration of receptor and donor catalytic reaction system is a key factor affecting HA sugar chain length, by measuring the chemical reaction system of donor / acceptor the molar concentration, the synthesis of uniform HA sugar chain controlled enzymatic method. Subsequently, the application of the homogeneous HA enzyme synthesis strategy in the preparation of HA conjugates with clear chemical structure and controlled HA chain length was further expanded. The biotin labeled hyaluronic acid (HA-biotin conjugates) and hyaluronic acid drug conjugates (HA-oroxylin conjugates) were prepared. HA-biotin conjugates can be used as a molecular probe to visualize the metabolic process of exogenous HA. HA-oroxylin conjugates as a model drug lays the foundation for the development of HA targeted anticancer drug. In the third chapter, the synthesis of hyaluronic acid - chondroitin sulfate (HA-CS) chimeric glycosaminoglycan was developed. Due to the similarity of HA and CS structure and PmHAS flexible substrate adaptability, four HA-CS chimeric glycosaminoglycans, CS2HA2-N3, HA2CS2HA2-N3, CS2HA4-N3 and CS4HA4-N3, were synthesized by stepwise reaction method, which was used to study the effect of microstructure change on HA biological function. This method can also be used in the synthesis of other different chimeric glycosaminoglycan, which lays a foundation for the study of the biological function of chimeric glycosaminoglycan. In the fourth part of the paper, combined with the work of our lab in the field of nucleotides synthesis, a HA synthesis strategy based on monosaccharide was established. Using the cheap monosaccharide GlcNAc and GlcA as the starting substrate, the enzymatic synthesis of sugar nucleotides was coupled with the polymerization of hyaluronic acid, and the one pot multi enzyme catalyzed and highly efficient synthesis of HA. The HA derivatives have been successfully synthesized by this method, and are used for the preparation and application of crosslinked HA, which has a very broad application prospect.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ281

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