阳离子化多糖基因传递载体的构建及其构效关系的研究

发布时间：2018-04-25 19:42

本文选题：阳离子多糖 + 乙二胺　；参考：《江苏大学》2017年硕士论文

【摘要】：天然多糖为自然界中广泛存在的纤维素及其衍生物,如几丁质、菊糖及肝素等天然高分子材料。天然多糖在生物体内具有重要作用,它不仅是生物体结构的主要组成成分之一,而且具有生物分子识别和信息传递的功能。目前关于天然多糖的生物功能报道非常多,如免疫调节、抗肿瘤、抗衰老和抗病毒等药理作用,但天然多糖作为基因载体的研究才刚刚起步,而关于其作为基因载体的构效关系的研究少之又少。本文选择杏鲍菇和金针菇两种食用真菌菇作为天然多糖的来源,分离、纯化得到三种精制多糖,分析其初级和高级结构,然后将乙二胺和精胺两种氨基化合物通过化学反应键合到天然多糖骨架上,使其带正电荷,具有压缩或者包裹基因的能力。依据阳离子多糖之间的转染效率差异性来初步阐述天然多糖的构效关系。第一章天然多糖在基因传递中的应用和研究进展安全高效的基因传递系统的开发是使基因治疗或DNA疫苗成为现实的主要挑战。天然多糖作为基因载体具有良好的转染效率,同时具有良好的生物相容性、生物可降解性和生物活性等优势,是一种非常有前景的基因载体材料。本章主要对天然多糖的分类及其阳离子进行总结,同时对其作为基因载体的应用和最新研究进展进行综述,并展望其应用前景和未来发展趋势。第二章天然多糖的提取、分离和纯化本章采用水作为提取溶剂,回流提取杏鲍菇和金针菇。采用乙醇沉淀和三氯乙酸除蛋白得到粗多糖,采用大孔树脂D101、纤维树脂DEAE-52、葡聚糖凝胶柱G100纯化,分别从杏鲍菇纯化得到精制多糖PEP,从金针菇纯化得到精制多糖FVP-D和FVP-X。对三种精制多糖进行分子量、单糖组分和单糖间连接方式和单糖构型研究,采用刚果红和原子力显微镜(AFM)进行高级结构分析。结果显示,PEP的平均相对分子量为1561.907 KDa,单糖组分主要为葡萄糖、半乳糖和甘露糖,摩尔比为8.76:1.97:1,三种单糖组分均为α构型,单糖间链连接方式主要为1→2或l→4位连接,刚果红和AFM实验表明PEP为球形而无螺旋结构的α-葡聚杂糖。FVP-D的平均相对分子量为198.198 KDa,单糖组分主要为半乳糖、葡萄糖和阿拉伯糖,摩尔比为51.36:2.37:15.55,单糖间链连接方式主要为1→3位连接,刚果红和afm实验表明fvp-d为棒状或球状的具有螺旋结构的阿拉伯糖-β-半乳聚糖。fvp-x的平均相对分子量为9.104kda,单糖组分主要为半乳糖、葡萄糖和甘露糖,摩尔比为1:14.37:0.83,单糖间链连接方式主要为1→2或l→4位连接,刚果红和afm实验表明fvp-x为单股螺旋链状的α-葡聚杂糖。第三章天然多糖的阳离子化修饰及其表征本章采用n,n-羰基二咪唑为连接剂,通过化学修饰的方法将乙二胺或者精胺键合到三种天然多糖骨架,制备得到六种阳离子多糖,采用红外、核磁和zeta电位测定对阳离子多糖进行表征,并分析阳离子多糖的单糖组分。结果显示,成功制备得到六种阳离子多糖,分别命名为pep-y、pep-j、fvp-d-y、fvp-d-j、fvp-x-y和fvp-x-j。六种阳离子多糖的zeta电位存在一定的差异性,其中pep-j的zeta电位最大,为45.8±0.97mv。fvp-x-y的zeta电位最小,为21.6±0.2mv。单糖组分结果显示,阳离子多糖保留了部分未被修饰的单糖组分。第四章阳离子多糖基因载体传递的构建本章将六种阳离子多糖通过静电作用与质粒sox2结合,形成阳离子多糖/psox2复合物,采用琼脂糖凝胶电泳对阳离子多糖的质粒滞留效果进行评价,采用动态光散射和zeta电位仪对阳离子多糖/psox2复合物进行粒径和zeta电位测定,采用afm对阳离子多糖/sox2复合物进行形态观察。结果显示,阳离子多糖/pdna复合物均能完全滞留质粒sox2,其最佳滞留比存在一定差异性。pep-y/psox2、pep-j/psox2、fvp-d-y/psox2和fvp-d-j/psox2复合物均为类球形的纳米粒,形态较规整。而fvp-x-y/psox2和fvp-x-j/psox2复合物的形态不规则,可能是因为其本身结构为单股螺旋链,且分子量较小,故与质粒dna结合后无法聚集成球状。第五章阳离子多糖/psox2复合物的转染效率及其机理研究本章采用mtt法对不同质量比的阳离子多糖/psox2复合物的细胞毒性进行考察,采用酶联免疫吸附测定法(elisa)和实时荧光定量pcr法(qrt-pcr)评价纳米粒对大鼠胚胎成纤维细胞(REF)的转染效率。以绿色荧光染料YOYO-1标记SOX2质粒,采用荧光显微镜研究阳离子多糖/pSOX2复合物的细胞摄取机理。结果显示,不同质量比的阳离子多糖/pSOX2复合物的毒性均明显低于阳性转染试剂Lipofectamine2000和PEI 25 KDa,其中PEP-J/pSOX2复合物的毒性相对最大。六种阳离子多糖/pSOX2复合物的转染效率比较高低如下: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)。且转染效率均高于Lipofectamine2000和PEI 25 KDa。构效关系初步分析如下:大分子量的、类球形的α-葡聚杂糖的转染效率高于中分子量的、棒状或者球状的β-阿拉伯-半乳聚糖,而后者的转染效率又高于低分子量的、单链状的α-葡聚杂糖。对两种高转染效率的PEP-J/pSOX2(40:1)和FVP-D-J/pSOX2(2:1)复合物的细胞摄取机理研究结果显示,PEP-J/pSOX2(40:1)主要是通过网格蛋白介导的内吞作用和小窝蛋白介导的内吞作用被细胞摄取,随即进入内涵体-溶酶体途经,在中间纤维蛋白和动力蛋白的参与作用下进行胞内转运,而FVP-D-J/pSOX2(2:1)复合物是通过中间纤维蛋白及其细胞骨架中的微丝和微管蛋白的参与下进行胞内转运。以上结果说明不同阳离子多糖其转染效率和细胞摄取机理存在一定的差异性。
[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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450

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