發(fā)布時間：2018-07-01 17:51

  本文選題：基因治療 + 殼聚糖改性　； 參考：《中國科學技術大學》2016年博士論文

【摘要】：目前,基因治療作為一項重要的醫(yī)療技術,已經(jīng)逐漸地被廣泛接受�；蛑委煵煌谝话愕尼t(yī)療手段,它立足于基因水平,是一種針對性很強、效果很好、微創(chuàng)性的醫(yī)療方法,尤其是對由于基因缺陷或者基因鏈段缺失等基因問題所引發(fā)的病癥。基因治療出現(xiàn)于1967年,此后一直以相當快的速度進行發(fā)展。鑒于它明顯的優(yōu)勢和廣闊的應用前景,也受到科學界越來越多的關注和重視。目前基因治療最主要的途徑是將完整的目的基因,通過基因運輸過程,帶入特定細胞中,從而補全缺失的或者取代有缺陷部分的基因片段,并進行后續(xù)正常的轉(zhuǎn)染過程,達到治療疾病的目的。而在這樣的整個基因治療中,最重要的也是最能決定治療效果的步驟,就是如何把目的基因帶進細胞體內(nèi),而這也是基因治療的難點。目前公認最合適的基因運輸手段,是通過基因載體。因此,能否獲得理想的基因載體,關系著基因治療的應用效果,以及未來基因治療的前景。本文的主要研究方向,就是制備理想的基因載體。基因治療傳統(tǒng)的載體是病毒,以及陽離子脂質(zhì)體或陽離子聚合物。盡管這些材料轉(zhuǎn)染效率高,但細胞毒性也很大,無法真正應用于臨床的基因治療。而自然界的多糖殼聚糖生物相容性好,對細胞無毒,完全可以用作基因載體,然而,殼聚糖的轉(zhuǎn)染效率太低。本論文以制備理想的基因載體為目的,通過常用殼聚糖的改性,賦予殼聚糖特定功能,提升殼聚糖的轉(zhuǎn)染效率。內(nèi)容包括：制備殼聚糖核殼結(jié)構進行基因緩釋；采用改性的殼聚糖制備功能性核殼結(jié)構納米粒子,用于基因轉(zhuǎn)染；采用60Co丫-射線輻射裂解法對殼聚糖大分子進行裂解,制備低分子量且轉(zhuǎn)染效率高的殼聚糖；采用輻射接枝法制備電荷密度大的接枝聚苯乙烯膦鹽的殼聚糖。主要研究內(nèi)容及成果如下：一、首先通過殼聚糖化學改性,制備巰基化烷基化殼聚糖(TACS)以及羥丁基殼聚糖(HBC),然后利用電荷吸附,讓TACS吸附基因質(zhì)粒,形成核粒子,再把HBC包裹在核粒子表面,制備殼聚糖核殼結(jié)構復合粒子。透射電子顯微鏡(TEM)研究表明,核殼粒子的粒徑約為120 nm。而且,粒子對細胞幾乎沒有毒性,細胞存活率在95%以上。同時,HEK 293T和Hela細胞體外緩釋以及轉(zhuǎn)染實驗表明,該核殼結(jié)構粒子可以有效達到緩釋的目的,轉(zhuǎn)染效率也比較高,達到了38.99%。二、利用聚乙二醇(PEG)對HBC進行進一步修飾,獲得EG-HBC,然后將EG-HBC包裹在吸附了基因質(zhì)粒的TACS核粒子表面,形成了TACS@EG-HBC核殼結(jié)構復合粒子。通過納米顆粒分析儀檢測,TACS@EG-HBC粒徑約為200 nm。之后,通過一系列的Hela細胞體外轉(zhuǎn)染和緩釋實驗,以及與TACS核粒子和TACS@HBC對比,證實了TACS@EG-HBC良好的緩釋功能和轉(zhuǎn)染能力。最后,在KM小鼠的體內(nèi)實驗中,驗證了TACS@EG-HBC轉(zhuǎn)染效果可以持續(xù)到60天,并且體內(nèi)的轉(zhuǎn)染效果很好。三、利用60Co γ-射線輻射裂解法,對殼聚糖溶液進行輻照,制備了低分子量的殼聚糖。通過改變吸收劑量,控制殼聚糖的分子量。烏氏粘度法測定表明,殼聚糖的分子量隨吸收劑量增大而降低,當吸收劑量從0到50 kGy變化時,分子量從35萬降低到5萬左右。動態(tài)光散射研究表明,輻照后的殼聚糖負載基因形成的粒子,其zeta電勢和粒徑都隨著吸收劑量的增大而減小。Hela細胞轉(zhuǎn)染實驗、熒光顯微鏡觀測以及流式細胞測定結(jié)果都表明,轉(zhuǎn)染效率有所提升,從4.76%最多提高到了11.1%。四、采用輻射接枝技術,將苯乙烯膦鹽接枝在殼聚糖上,制備出殼聚糖接枝聚苯乙烯膦鹽的接枝物(CS-P)。13C-NMR表征顯示,苯環(huán)與P原子同時出現(xiàn)在殼聚糖上,證明CS-P的成功制備。熱重分析(TG)分析得到接枝率為2.51%。DLS研究結(jié)果表明,改性后的殼聚糖形成的粒子的zeta電勢要比未改性的高很多。細胞毒性實驗表明,接枝物對細胞無毒,Hela細胞存活率達到93.1±1.61%。同時Hela細胞的體外轉(zhuǎn)染實驗表明,基因轉(zhuǎn)染效率得到了較大提升,從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%.
【學位授予單位】：中國科學技術大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R450;O636.1

【相似文獻】

相關期刊論文 前1條

1 稻角忠弘 ,許晉初;燒結(jié)過程最佳風量全面控制系統(tǒng)(TACS)的研究與應用[J];燒結(jié)球團;1987年03期

相關重要報紙文章 前1條

1 湖北棗陽移動通信公司 劉斌;談數(shù)字化莫入誤區(qū)[N];人民日報;2000年

相關博士學位論文 前1條

1 林福星;改性殼聚糖制備及其基因轉(zhuǎn)染應用研究[D];中國科學技術大學;2016年

相關碩士學位論文 前1條

1 王云;羥丁基殼聚糖—巰基烷基化殼聚糖/質(zhì)粒殼核結(jié)構的制備及其在體內(nèi)外緩釋基因的研究[D];安徽醫(yī)科大學;2014年

，

本文編號：2088587