發(fā)布時(shí)間：2018-04-11 10:05

  本文選題：非病毒基因治療載體 + PAMAM�。� 參考：《天津大學(xué)》2006年碩士論文

【摘要】： 在基因治療的過(guò)程中,基因載體有著極其重要的作用。對(duì)于常用的陽(yáng)離子聚合物/質(zhì)粒DNA非病毒基因治療載體來(lái)講,其與質(zhì)粒DNA之間的相互作用包括靜電,疏水等。而在基因傳遞的過(guò)程中,疏水作用究竟起一個(gè)什么樣的作用是一個(gè)受到重視的問(wèn)題。本文研究的主要內(nèi)容有: 首先,本文參考文獻(xiàn),在液相中對(duì)PAMAM(polyamidoamine)進(jìn)行了表面修飾,為了體現(xiàn)疏水性的作用,我們選擇了帶有苯環(huán)的苯丙氨酸(Phe)和色氨酸(Trp)作為表面修飾物。核磁共振檢測(cè)證明了PAMAM表面連接了足夠的氨基酸殘基,生成了表面既有氨基(在水溶液中會(huì)帶正電荷)又有大量疏水基的PAMAM氨基酸修飾物。 采用了質(zhì)粒提取試劑盒,色譜法和聚乙二醇法提取了得到實(shí)驗(yàn)所需的質(zhì)粒,并利用電泳和紫外吸收來(lái)檢測(cè)了質(zhì)粒的質(zhì)量和濃度,使之能夠用于后續(xù)的實(shí)驗(yàn)。 為了考查新聚合物和質(zhì)粒形成的復(fù)合物的自聚過(guò)程,瓊脂糖凝膠電泳,溴乙錠熒光發(fā)射,多角度激光散射和透射電鏡從不同的方面顯示了聚合物和質(zhì)粒DNA之間的作用和質(zhì)粒的縮合。 聚合物對(duì)于動(dòng)物細(xì)胞的毒性實(shí)驗(yàn)和轉(zhuǎn)染效率實(shí)驗(yàn)反應(yīng)了新聚合物的生物毒性和最終的轉(zhuǎn)染效率。在低的濃度下,聚合物有相對(duì)較弱的毒性,在電荷比例為5的時(shí)候,修飾Phe和Trp的聚合物能夠大大提高綠色熒光蛋白在非洲綠猿腎細(xì)胞中的表達(dá)效率。 最終說(shuō)明,含疏水基的氨基酸在PAMAM表面的引入,改變了PAMAM和質(zhì)粒DNA之間的自聚過(guò)程,并最終提高了細(xì)胞轉(zhuǎn)染的效率,說(shuō)明了疏水作用在基因傳遞系統(tǒng)中的重要作用,為陽(yáng)離子聚合物基因載體今后的發(fā)展提供了一個(gè)重要的參考
[Abstract]:Gene vectors play an important role in gene therapy.For the commonly used cationic polymer / plasmid DNA non-viral gene therapy vector, its interaction with plasmid DNA includes electrostatic, hydrophobic, and so on.In the process of gene transmission, what kind of hydrophobic effect is paid attention to.The main contents of this paper are as follows:Firstly, the surface modification of PamAmidoamine was carried out in liquid phase with reference. In order to show the hydrophobicity, the phenylalanine (Phe) and tryptophan (Trp) with benzene ring were selected as the surface modifiers.Nuclear magnetic resonance (NMR) results show that the surface of PAMAM has enough amino acid residues to form PAMAM amino acid modifiers with both amino acids (which are positively charged in aqueous solution) and hydrophobic groups.The plasmids were extracted by using plasmid extraction kit chromatography and polyethylene glycol method. The quality and concentration of plasmids were determined by electrophoresis and UV absorption so that the plasmids could be used in subsequent experiments.In order to investigate the self-polymerization process of the complexes formed by new polymers and plasmids, agarose gel electrophoresis, fluorescence emission of bromoethidium,Multi-angle laser scattering and transmission electron microscopy show the interaction between polymer and plasmid DNA and the condensation of plasmids.The toxicity test and transfection efficiency experiment of polymer to animal cells reflect the biological toxicity and the final transfection efficiency of the new polymer.At a low concentration, the polymer has relatively weak toxicity. When the charge ratio is 5, the polymer modified with Phe and Trp can greatly improve the expression efficiency of green fluorescent protein in the kidney cells of the African ape.Finally, the introduction of hydrophobic amino acids on the surface of PAMAM changed the self-polymerization process between PAMAM and plasmid DNA, and finally improved the efficiency of cell transfection, which indicated the important role of hydrophobic action in gene transmission system.It provides an important reference for the future development of cationic polymer gene vector.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王曉婷;朱蔭昌;高秋端;郭玲香;李新松;;日本血吸蟲(chóng)病新型樹(shù)枝狀載體-DNA疫苗的構(gòu)建及其評(píng)價(jià)[J];中國(guó)血吸蟲(chóng)病防治雜志;2010年01期

相關(guān)碩士學(xué)位論文 前1條

1 王瑩;膽固醇在PAMAM介導(dǎo)的基因傳遞過(guò)程中的作用[D];天津大學(xué);2008年

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本文編號(hào)：1735525