改性壳聚糖制备及其基因转染应用研究

发布时间：2018-07-01 17:51

本文选题：基因治疗 + 壳聚糖改性　；参考：《中国科学技术大学》2016年博士论文

【摘要】：目前,基因治疗作为一项重要的医疗技术,已经逐渐地被广泛接受。基因治疗不同于一般的医疗手段,它立足于基因水平,是一种针对性很强、效果很好、微创性的医疗方法,尤其是对由于基因缺陷或者基因链段缺失等基因问题所引发的病症。基因治疗出现于1967年,此后一直以相当快的速度进行发展。鉴于它明显的优势和广阔的应用前景,也受到科学界越来越多的关注和重视。目前基因治疗最主要的途径是将完整的目的基因,通过基因运输过程,带入特定细胞中,从而补全缺失的或者取代有缺陷部分的基因片段,并进行后续正常的转染过程,达到治疗疾病的目的。而在这样的整个基因治疗中,最重要的也是最能决定治疗效果的步骤,就是如何把目的基因带进细胞体内,而这也是基因治疗的难点。目前公认最合适的基因运输手段,是通过基因载体。因此,能否获得理想的基因载体,关系着基因治疗的应用效果,以及未来基因治疗的前景。本文的主要研究方向,就是制备理想的基因载体。基因治疗传统的载体是病毒,以及阳离子脂质体或阳离子聚合物。尽管这些材料转染效率高,但细胞毒性也很大,无法真正应用于临床的基因治疗。而自然界的多糖壳聚糖生物相容性好,对细胞无毒,完全可以用作基因载体,然而,壳聚糖的转染效率太低。本论文以制备理想的基因载体为目的,通过常用壳聚糖的改性,赋予壳聚糖特定功能,提升壳聚糖的转染效率。内容包括：制备壳聚糖核壳结构进行基因缓释；采用改性的壳聚糖制备功能性核壳结构纳米粒子,用于基因转染；采用60Co丫-射线辐射裂解法对壳聚糖大分子进行裂解,制备低分子量且转染效率高的壳聚糖；采用辐射接枝法制备电荷密度大的接枝聚苯乙烯膦盐的壳聚糖。主要研究内容及成果如下：一、首先通过壳聚糖化学改性,制备巯基化烷基化壳聚糖(TACS)以及羟丁基壳聚糖(HBC),然后利用电荷吸附,让TACS吸附基因质粒,形成核粒子,再把HBC包裹在核粒子表面,制备壳聚糖核壳结构复合粒子。透射电子显微镜(TEM)研究表明,核壳粒子的粒径约为120 nm。而且,粒子对细胞几乎没有毒性,细胞存活率在95%以上。同时,HEK 293T和Hela细胞体外缓释以及转染实验表明,该核壳结构粒子可以有效达到缓释的目的,转染效率也比较高,达到了38.99%。二、利用聚乙二醇(PEG)对HBC进行进一步修饰,获得EG-HBC,然后将EG-HBC包裹在吸附了基因质粒的TACS核粒子表面,形成了TACS@EG-HBC核壳结构复合粒子。通过纳米颗粒分析仪检测,TACS@EG-HBC粒径约为200 nm。之后,通过一系列的Hela细胞体外转染和缓释实验,以及与TACS核粒子和TACS@HBC对比,证实了TACS@EG-HBC良好的缓释功能和转染能力。最后,在KM小鼠的体内实验中,验证了TACS@EG-HBC转染效果可以持续到60天,并且体内的转染效果很好。三、利用60Co γ-射线辐射裂解法,对壳聚糖溶液进行辐照,制备了低分子量的壳聚糖。通过改变吸收剂量,控制壳聚糖的分子量。乌氏粘度法测定表明,壳聚糖的分子量随吸收剂量增大而降低,当吸收剂量从0到50 kGy变化时,分子量从35万降低到5万左右。动态光散射研究表明,辐照后的壳聚糖负载基因形成的粒子,其zeta电势和粒径都随着吸收剂量的增大而减小。Hela细胞转染实验、荧光显微镜观测以及流式细胞测定结果都表明,转染效率有所提升,从4.76%最多提高到了11.1%。四、采用辐射接枝技术,将苯乙烯膦盐接枝在壳聚糖上,制备出壳聚糖接枝聚苯乙烯膦盐的接枝物(CS-P)。13C-NMR表征显示,苯环与P原子同时出现在壳聚糖上,证明CS-P的成功制备。热重分析(TG)分析得到接枝率为2.51%。DLS研究结果表明,改性后的壳聚糖形成的粒子的zeta电势要比未改性的高很多。细胞毒性实验表明,接枝物对细胞无毒,Hela细胞存活率达到93.1±1.61%。同时Hela细胞的体外转染实验表明,基因转染效率得到了较大提升,从4.59%提高到了32.8%。
[Abstract]:Gene therapy is now widely accepted as an important medical technology. Gene therapy is different from general medical treatment. It is based on the gene level. It is a highly targeted, very effective, minimally invasive medical method, especially the disease caused by genetic defects or deletion of gene chain segments. Gene therapy has been developed in 1967 and has been developing at a very fast speed since then. In view of its obvious advantages and broad application prospects, more and more attention and attention have been paid by the scientific community. The main way for gene therapy is to bring the complete target gene into specific cells through the process of gene transport. The most important and most important step in the whole gene therapy is how to bring the target gene into the cell, which is the difficult point of the gene therapy. The most suitable gene transport means through gene carriers. Therefore, the possibility of obtaining an ideal gene carrier is related to the application of gene therapy and the prospect of future gene therapy. The main research direction in this paper is to prepare an ideal gene carrier. The carrier of gene therapy is the virus, and the cationic liposome or yang. Although these materials have high transfection efficiency, but they are very toxic and can not be used in clinical gene therapy, the natural polysaccharide chitosan has good biocompatibility, no toxic to cells and can be used as a gene carrier. However, the efficiency of chitosan is too low. This paper aims to prepare an ideal gene carrier. To improve the transfection efficiency of chitosan through chitosan modification, chitosan has been given specific functions to improve the transfection efficiency of chitosan. The contents include: preparing chitosan core and shell structure for gene release, using modified chitosan to prepare functional nuclear shell nanoparticles for gene transfection, and using 60Co Ya ray radiolysis method for chitosan macromolecules To prepare chitosan with low molecular weight and high transfection efficiency, the grafting of polystyrene phosphonium salt with large charge density was prepared by radiation grafting. The main contents and results were as follows: first, chitosan (TACS) and hydroxybutyl chitosan (HBC) were prepared by chemical modification of chitosan. With a charge adsorption, TACS can adsorb gene plasmids and form nuclear particles and then encapsulate HBC on the surface of the nuclear particles to prepare the chitosan core shell composite particles. The transmission electron microscope (TEM) study shows that the particle size of the core shell particles is about 120 nm. and the particles have little toxicity to the cells and the cell survival rate is above 95%. Meanwhile, HEK 293T and Hela are fine. The slow-release and transfection experiment in vitro showed that the core shell structure particles could be effective for sustained release, and the transfection efficiency was also high, reaching 38.99%. two. HBC was further modified by polyethylene glycol (PEG), and EG-HBC was obtained. Then EG-HBC was wrapped on the surface of TACS nuclear particles that adsorbed the gene plasmids, forming a TACS@EG-HBC nuclear shell. After the particle size analyzer was detected by the nano particle analyzer, after the particle size of TACS@EG-HBC was about 200 nm., a series of Hela cells were transfected and released in vitro, and compared with TACS nuclear particles and TACS@HBC, the good release function and transfection ability of TACS@EG-HBC were confirmed. The most later, TACS@EG-HB was verified in the vivo experiment of KM mice. The transfection effect of C can last to 60 days, and the transfection effect in the body is very good. Three, the chitosan solution is irradiated by 60Co gamma ray radiation, and the chitosan with low molecular weight is prepared. The molecular weight of chitosan is controlled by changing the absorbed dose. The molecular weight of chitosan is increased with the absorption dose. When the absorbed dose was changed from 0 to 50 kGy, the molecular weight decreased from 350 thousand to about 50 thousand. The dynamic light scattering study showed that the zeta potential and particle size of the chitosan loaded gene after irradiation decreased with the increase of the absorbed dose, and the.Hela cell transfection experiment was reduced, the fluorescence microscope observation and the flow cytometry results were all. The results showed that the transfection efficiency increased from 4.76% to 11.1%. four. The graft copolymer of styrene phosphine salt was grafted onto chitosan by radiation grafting technique, and the graft copolymer of polystyrene phosphine salts (CS-P).13C-NMR was prepared by graft copolymerization of chitosan grafted polystyrene phosphine salt, which showed that the benzene ring and P atoms were produced at the same time with the chitosan, which proved that the CS-P was successfully prepared. Thermo gravimetric analysis (TG). The results of the analysis of the grafting ratio of 2.51%.DLS showed that the zeta potential of the particles formed by the modified chitosan was much higher than that of the unmodified. The cytotoxicity test showed that the graft was non-toxic to the cells, the survival rate of Hela cells reached 93.1 + 1.61%. and the transfer experiment of Hela cells in vitro showed that the gene transfection efficiency was greatly raised. Up, up from 4.59% to 32.8%.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R450;O636.1

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