本文選題：陽(yáng)離子多糖 + 乙二胺�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：天然多糖為自然界中廣泛存在的纖維素及其衍生物,如幾丁質(zhì)、菊糖及肝素等天然高分子材料。天然多糖在生物體內(nèi)具有重要作用,它不僅是生物體結(jié)構(gòu)的主要組成成分之一,而且具有生物分子識(shí)別和信息傳遞的功能。目前關(guān)于天然多糖的生物功能報(bào)道非常多,如免疫調(diào)節(jié)、抗腫瘤、抗衰老和抗病毒等藥理作用,但天然多糖作為基因載體的研究才剛剛起步,而關(guān)于其作為基因載體的構(gòu)效關(guān)系的研究少之又少。本文選擇杏鮑菇和金針菇兩種食用真菌菇作為天然多糖的來(lái)源,分離、純化得到三種精制多糖,分析其初級(jí)和高級(jí)結(jié)構(gòu),然后將乙二胺和精胺兩種氨基化合物通過(guò)化學(xué)反應(yīng)鍵合到天然多糖骨架上,使其帶正電荷,具有壓縮或者包裹基因的能力。依據(jù)陽(yáng)離子多糖之間的轉(zhuǎn)染效率差異性來(lái)初步闡述天然多糖的構(gòu)效關(guān)系。第一章天然多糖在基因傳遞中的應(yīng)用和研究進(jìn)展安全高效的基因傳遞系統(tǒng)的開發(fā)是使基因治療或DNA疫苗成為現(xiàn)實(shí)的主要挑戰(zhàn)。天然多糖作為基因載體具有良好的轉(zhuǎn)染效率,同時(shí)具有良好的生物相容性、生物可降解性和生物活性等優(yōu)勢(shì),是一種非常有前景的基因載體材料。本章主要對(duì)天然多糖的分類及其陽(yáng)離子進(jìn)行總結(jié),同時(shí)對(duì)其作為基因載體的應(yīng)用和最新研究進(jìn)展進(jìn)行綜述,并展望其應(yīng)用前景和未來(lái)發(fā)展趨勢(shì)。第二章天然多糖的提取、分離和純化本章采用水作為提取溶劑,回流提取杏鮑菇和金針菇。采用乙醇沉淀和三氯乙酸除蛋白得到粗多糖,采用大孔樹脂D101、纖維樹脂DEAE-52、葡聚糖凝膠柱G100純化,分別從杏鮑菇純化得到精制多糖PEP,從金針菇純化得到精制多糖FVP-D和FVP-X。對(duì)三種精制多糖進(jìn)行分子量、單糖組分和單糖間連接方式和單糖構(gòu)型研究,采用剛果紅和原子力顯微鏡(AFM)進(jìn)行高級(jí)結(jié)構(gòu)分析。結(jié)果顯示,PEP的平均相對(duì)分子量為1561.907 KDa,單糖組分主要為葡萄糖、半乳糖和甘露糖,摩爾比為8.76:1.97:1,三種單糖組分均為α構(gòu)型,單糖間鏈連接方式主要為1→2或l→4位連接,剛果紅和AFM實(shí)驗(yàn)表明PEP為球形而無(wú)螺旋結(jié)構(gòu)的α-葡聚雜糖。FVP-D的平均相對(duì)分子量為198.198 KDa,單糖組分主要為半乳糖、葡萄糖和阿拉伯糖,摩爾比為51.36:2.37:15.55,單糖間鏈連接方式主要為1→3位連接,剛果紅和afm實(shí)驗(yàn)表明fvp-d為棒狀或球狀的具有螺旋結(jié)構(gòu)的阿拉伯糖-β-半乳聚糖。fvp-x的平均相對(duì)分子量為9.104kda,單糖組分主要為半乳糖、葡萄糖和甘露糖,摩爾比為1:14.37:0.83,單糖間鏈連接方式主要為1→2或l→4位連接,剛果紅和afm實(shí)驗(yàn)表明fvp-x為單股螺旋鏈狀的α-葡聚雜糖。第三章天然多糖的陽(yáng)離子化修飾及其表征本章采用n,n-羰基二咪唑?yàn)檫B接劑,通過(guò)化學(xué)修飾的方法將乙二胺或者精胺鍵合到三種天然多糖骨架,制備得到六種陽(yáng)離子多糖,采用紅外、核磁和zeta電位測(cè)定對(duì)陽(yáng)離子多糖進(jìn)行表征,并分析陽(yáng)離子多糖的單糖組分。結(jié)果顯示,成功制備得到六種陽(yáng)離子多糖,分別命名為pep-y、pep-j、fvp-d-y、fvp-d-j、fvp-x-y和fvp-x-j。六種陽(yáng)離子多糖的zeta電位存在一定的差異性,其中pep-j的zeta電位最大,為45.8±0.97mv。fvp-x-y的zeta電位最小,為21.6±0.2mv。單糖組分結(jié)果顯示,陽(yáng)離子多糖保留了部分未被修飾的單糖組分。第四章陽(yáng)離子多糖基因載體傳遞的構(gòu)建本章將六種陽(yáng)離子多糖通過(guò)靜電作用與質(zhì)粒sox2結(jié)合,形成陽(yáng)離子多糖/psox2復(fù)合物,采用瓊脂糖凝膠電泳對(duì)陽(yáng)離子多糖的質(zhì)粒滯留效果進(jìn)行評(píng)價(jià),采用動(dòng)態(tài)光散射和zeta電位儀對(duì)陽(yáng)離子多糖/psox2復(fù)合物進(jìn)行粒徑和zeta電位測(cè)定,采用afm對(duì)陽(yáng)離子多糖/sox2復(fù)合物進(jìn)行形態(tài)觀察。結(jié)果顯示,陽(yáng)離子多糖/pdna復(fù)合物均能完全滯留質(zhì)粒sox2,其最佳滯留比存在一定差異性。pep-y/psox2、pep-j/psox2、fvp-d-y/psox2和fvp-d-j/psox2復(fù)合物均為類球形的納米粒,形態(tài)較規(guī)整。而fvp-x-y/psox2和fvp-x-j/psox2復(fù)合物的形態(tài)不規(guī)則,可能是因?yàn)槠浔旧斫Y(jié)構(gòu)為單股螺旋鏈,且分子量較小,故與質(zhì)粒dna結(jié)合后無(wú)法聚集成球狀。第五章陽(yáng)離子多糖/psox2復(fù)合物的轉(zhuǎn)染效率及其機(jī)理研究本章采用mtt法對(duì)不同質(zhì)量比的陽(yáng)離子多糖/psox2復(fù)合物的細(xì)胞毒性進(jìn)行考察,采用酶聯(lián)免疫吸附測(cè)定法(elisa)和實(shí)時(shí)熒光定量pcr法(qrt-pcr)評(píng)價(jià)納米粒對(duì)大鼠胚胎成纖維細(xì)胞(REF)的轉(zhuǎn)染效率。以綠色熒光染料YOYO-1標(biāo)記SOX2質(zhì)粒,采用熒光顯微鏡研究陽(yáng)離子多糖/pSOX2復(fù)合物的細(xì)胞攝取機(jī)理。結(jié)果顯示,不同質(zhì)量比的陽(yáng)離子多糖/pSOX2復(fù)合物的毒性均明顯低于陽(yáng)性轉(zhuǎn)染試劑Lipofectamine2000和PEI 25 KDa,其中PEP-J/pSOX2復(fù)合物的毒性相對(duì)最大。六種陽(yáng)離子多糖/pSOX2復(fù)合物的轉(zhuǎn)染效率比較高低如下:PEP-J/pSOX2(40:1)FVP-D-J/pSOX2(40:1)PEP-Y/pSOX2(20:1)FVP-D-Y/SOX2(20:1)FVP-X-J/pSOX2(2:1)FVP-X-Y/SOX2(20:1)。且轉(zhuǎn)染效率均高于Lipofectamine2000和PEI 25 KDa。構(gòu)效關(guān)系初步分析如下:大分子量的、類球形的α-葡聚雜糖的轉(zhuǎn)染效率高于中分子量的、棒狀或者球狀的β-阿拉伯-半乳聚糖,而后者的轉(zhuǎn)染效率又高于低分子量的、單鏈狀的α-葡聚雜糖。對(duì)兩種高轉(zhuǎn)染效率的PEP-J/pSOX2(40:1)和FVP-D-J/pSOX2(2:1)復(fù)合物的細(xì)胞攝取機(jī)理研究結(jié)果顯示,PEP-J/pSOX2(40:1)主要是通過(guò)網(wǎng)格蛋白介導(dǎo)的內(nèi)吞作用和小窩蛋白介導(dǎo)的內(nèi)吞作用被細(xì)胞攝取,隨即進(jìn)入內(nèi)涵體-溶酶體途經(jīng),在中間纖維蛋白和動(dòng)力蛋白的參與作用下進(jìn)行胞內(nèi)轉(zhuǎn)運(yùn),而FVP-D-J/pSOX2(2:1)復(fù)合物是通過(guò)中間纖維蛋白及其細(xì)胞骨架中的微絲和微管蛋白的參與下進(jìn)行胞內(nèi)轉(zhuǎn)運(yùn)。以上結(jié)果說(shuō)明不同陽(yáng)離子多糖其轉(zhuǎn)染效率和細(xì)胞攝取機(jī)理存在一定的差異性。
[Abstract]:Natural polysaccharide is a kind of natural cellulose and its derivatives, such as chitin, chrysanthemum and heparin, such as chitin, chrysanthemum and heparin. Natural polysaccharide plays an important role in organism. It is not only one of the main components of organism structure, but also has the function of biological molecular recognition and information transmission. There are many reports on the biological function of sugar, such as immune regulation, anti-tumor, anti aging and antiviral action, but the study of natural polysaccharide as a gene carrier has just started, but there are few studies on its structure-activity relationship as a gene carrier. In this paper, two kinds of mushroom and mushroom mushroom were selected as natural polysaccharides. The source, separation and purification of three refined polysaccharides were obtained, and their primary and advanced structures were analyzed. Then the two amino compounds of ethylamine and spermine were bonded to the natural polysaccharide skeleton by chemical reaction to make them positive charge and have the ability to compress or encapsulate genes. The construction of natural polysaccharides. Chapter 1: the application and research of natural polysaccharide in gene transfer; development of safe and efficient gene transfer system is the main challenge to make gene therapy or DNA vaccine become a reality. Natural polysaccharide has good transfection efficiency as a gene carrier and has good biocompatibility at the same time. In this chapter, the classification and cations of natural polysaccharides are summarized, and the application and the latest research progress of the natural polysaccharides are summarized, and the application prospect and future development trend are also prospected. The second chapter is the extraction and separation of natural polysaccharides. In this chapter, water is used as extraction solvent and reflux extraction of Pleurotus abalone and Flammulina velutipes. Ethanol precipitation and three chloroacetic acid were used to get crude polysaccharide. Macroporous resin D101, fibrous resin DEAE-52 and glucan gel column G100 were used to purify the purified polysaccharide PEP from Pleurotus abalone. Purified polysaccharide FVP-D and FV were purified from Flammulina velutipes The molecular weight, monosaccharide and monosaccharide connection and monosaccharide configuration of three refined polysaccharides were studied by high level structure analysis using Congo red and atomic force microscope (AFM). The results showed that the average relative molecular weight of PEP was 1561.907 KDa, and the monosaccharide components were mainly glucose, galactose and mannose, and the molar ratio was 8.76:1.97:1, The three monosaccharide components were all alpha, and the main chain connections of monosaccharide were 1 to 2 or l to 4. Congo red and AFM experiments showed that the average relative molecular weight of PEP as spherical but without spiral structure was 198.198 KDa, and monosaccharide components were mainly galactose, grape sugar and Arabia sugar, and mole ratio 51.36:2.37:15.55, single The main carbohydrate chain connection is 1 to 3 connections. Congo red and AFM experiments show that the average relative molecular weight of the fvp-d sugar - beta galactan.Fvp-x with a rod like or ball like structure is 9.104kda. The monosaccharide components are mainly galactose, glucose and mannose, mmoli is 1:14.37:0.83, and the monosaccharide chain connection is mainly For 1 - 2 or L - 4 connections, Congo red and AFM experiments show that fvp-x is a single strand of spiral chain like alpha gluconate. Third chapter third natural polysaccharides cationic modification and characterization. This chapter uses n, n- carbonyl two imidazole as a connector, chemically modified ethylenediamine or spermine bond to three natural polysaccharides skeleton, prepared six species Cationic polysaccharides were characterized by IR, NMR and zeta potentials, and the monosaccharide components of cationic polysaccharides were analyzed. The results showed that six cationic polysaccharides were successfully prepared, named as pep-y, pep-j, fvp-d-y, fvp-d-j, fvp-x-y and fvp-x-j., and the zeta potential of six cationic polysaccharides was different. The zeta potential of pep-j was the largest and the zeta potential of 45.8 + 0.97mv.fvp-x-y was the smallest. The result of the 21.6 + 0.2mv. monosaccharide component showed that the cationic polysaccharide retained a part of the monosaccharide component which was not modified. The construction of the fourth chapter cationic polysaccharide gene carrier was formed by combining the electrostatic interaction with the plasmid Sox2 to form the cationic polysaccharide. The cationic polysaccharide /psox2 complex was evaluated by agarose gel electrophoresis. The particle size and zeta potential of cationic polysaccharide /psox2 complex were measured by dynamic light scattering and zeta potentiometer. The morphology of cationic polyose /sox2 complex was observed by AFM. The sugar /pdna complex can all stay plasmid Sox2 completely, and its optimal retention ratio exists a certain difference.Pep-y/psox2, pep-j/psox2, fvp-d-y/psox2 and fvp-d-j/psox2 complex are all spherical nanoparticles, and the morphology is more regular, but the morphology of fvp-x-y/psox2 and fvp-x-j/psox2 complex is irregular, probably because its structure is single strand helix. Chain, and the molecular weight is small, so it can not integrate with plasmid DNA. Fifth the transfection efficiency of cationic polysaccharide /psox2 complex and its mechanism study the cytotoxicity of the cationic polysaccharide /psox2 complex with different mass ratio by MTT method. The enzyme linked immunosorbent assay (ELISA) and real time fluorescence quantification are used. The transfection efficiency of nanoparticles to rat embryonic fibroblasts (REF) was evaluated by PCR method (qRT-PCR). The cell uptake mechanism of cationic polysaccharide /pSOX2 complex was studied by fluorescent dye YOYO-1 labeled SOX2 plasmid. The results showed that the toxicity of the cationic polysaccharide /pSOX2 complex with different mass ratio was significantly lower than that of the positive transformation. The toxicity of Lipofectamine2000 and PEI 25 KDa was the largest. The transfection efficiency of the six cationic polysaccharides /pSOX2 complex was as follows: PEP-J/pSOX2 (40:1) FVP-D-J/pSOX2 (40:1) PEP-Y/pSOX2 (20:1) FVP-D-Y/SOX2 (20:1). The preliminary analysis of the structure-activity relationship between mine2000 and PEI 25 KDa. is as follows: the transfection efficiency of large molecular weight, spherical alpha gluconi is higher than that of medium molecular weight, rod like or spherical beta Arabia galactan, and the latter is more efficient than low molecular weight, single chain alpha glucoheterose. Two kinds of high transfection efficiency PEP-J/pSOX2 (40: 1) the study of cell uptake mechanism of FVP-D-J/pSOX2 (2:1) complex shows that PEP-J/pSOX2 (40:1) is mainly absorbed by the endocytosis and endocytosis mediated by fossa protein, and then enters the endosome lysosome, and carries out intracellular transport under the participation of intermediate fibrinolytic and kinetic protein. The FVP-D-J/pSOX2 (2:1) complex is transported within the cell through the involvement of microfilament and microtubulin in the intermediate fibrin and its cytoskeleton. The results show that there is a certain difference in the transfection efficiency and the cell uptake mechanism of different cationic polysaccharides.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R450

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