PAMAM-HA靶向基因药物传递系统构建及生物性能的评价

发布时间：2018-07-09 17:17

本文选题：基因载体 + PAMAM　；参考：《广东药学院》2015年硕士论文

【摘要】：基因治疗是通过一定的方法、方式,将外源基因导入目的细胞并使其有效表达,以纠正基因的缺陷或在细胞中发挥作用,从而发挥治疗疾病的作用。基因治疗的关键就是选择合适的载体将目的基因准确的运送到靶细胞,并控制使其有效表达,发挥治疗作用。病毒型载体具有较高的转染效率,但病毒载体存在较严重的安全性问题。非病毒载体主要有阳离子聚合物、阳离子多肽和阳离子脂质体等,具有靶向性,低细胞毒性,较高的安全性,并且易于进行结构修饰等优点,已成为基因治疗中的热点,逐渐受到了越来越多的关注,对其研究已逐渐深入。PAMAM具有内部空腔结构,其末端基团精确可控,氨基易质子化而带正电,容易与DNA结合形成纳米级复合物,可以保护DNA并达到转染的目的,作为药物载体和基因载体具有较高的稳定性、生物兼容性良好、无免疫源性、使用剂量下低细胞毒性的优势。对PAMAM树状大分子末端基团进行表面修饰,减少表面的正电荷数目,以降低其细胞毒性,改善基因转染效率。透明质酸(Hyaluronic,简称HA)作为天然水溶性高分子,具有多种适宜作为作为药物载体的优良特性,包括生物兼容性良好、非免疫原性、高生物活性及易被体内酶作用而天然降解、靶向性。本研究以HA具有较好的肿瘤靶向性、增强复合物水溶性、生物兼容性,PAMAM具有较好的携带目的基因导入细胞中的理论基础为切入点,拟构建HA-PAMAM/p EGFP基因传递表达系统,深入研究其物理化学性能、生物性能、靶向性等方面的影响;从PAMAM分子量,HA接枝率和接枝密度,聚阳离子/基因重量比(w/w)等方面优化该传递系统,提高基因转染效率、降低细胞毒性、提高载体靶向性、稳定性等。本研究的主要内容:(1)PAMAM-HA聚合物的化学合成和结构表征:采用透明质酸(HA)与PAMAM在含有Na Cl的四氢硼酸钠(Na BH4)缓冲液中(p H8.5),在氰硼氢化纳(Na BH3CN)催化下反应生成PAMA-HA聚合物。不同PAMAM分子量、HA接枝量和接枝密度的PAMAM-HA样品同步制备并作比较。PAMAM-HA样品的化学结构有红外(IR)等鉴定。(2)对PAMAM-HA作为基因载体的性能考察:采用动态光散射发测定了PAMAM-HA/DNA复合物的粒径分布及表面电位;采用透射电镜(TEM)观察复合物粒子形态;通过琼脂糖凝胶阻滞实验考察PAMAM-HA结合并压缩DNA的能力。(3)PAMAM-HA细胞毒性:用MTT法考察PAMAM-HA各样品对不同肿瘤细胞的细胞毒性,并考察在PAMAM-HA/DNA的转染比例下的毒性。(4)PAMAM-HA体外转染研究:用Bel-7402细胞对PAMAM-HA/DNA复合物的最佳转染效率进行筛选并考察PAMAM-HA/DNA复合物在不同细胞之间的转染与PEI25K相比较,同时考察血清对转染效率的影响。(5)PAMAM-HA诱导细胞凋亡:用Annexin V-FITC细胞凋亡法考察PAMAM-HA各样品在在实验浓度下是否可诱导细胞凋亡及凋亡效率与浓度和时间的关系。(6)PAMAM-HA转染机制研究:用不同摄取途径抑制剂考察各复合物进入细胞的途径,将细胞核、溶酶体和DNA分别染色标记用激光共聚焦显微镜观察考察DNA在细胞内的分布。
[Abstract]:Gene therapy is a way of introducing foreign genes into the target cells and making them effective in order to correct the defects of the genes or to play a role in the cells. The key to gene therapy is to select the appropriate carrier to deliver the target base to the target cell accurately and control it to be effective. Viral vectors have high transfection efficiency, but viral vectors have serious safety problems. Non viral vectors mainly have cationic polymers, cationic polypeptides and cationic liposomes, which have the advantages of targeting, low cytotoxicity, high safety, and easy to carry out structural modification. More and more attention has been paid to the hot spots in gene therapy. The research has gradually deepened.PAMAM with the internal cavity structure, its terminal group is precise and controllable, the amino group is easy to protonated and positively charged, and it is easy to combine with DNA to form nano scale complex. It can protect DNA and reach the purpose of transfection as drug carrier and gene carrier. With the advantages of high stability, good biocompatibility, no immunogenic and low cytotoxicity at the dosage, surface modification of the terminal group of PAMAM tree macromolecules to reduce the number of positive charges on the surface in order to reduce its cytotoxicity and improve gene transfection efficiency. Hyaluronic (HA) as a natural water-soluble high score It has a variety of excellent properties as a drug carrier, including good biocompatibility, non immunogenicity, high bioactivity and easy to be degraded naturally by enzymes in the body. The HA has good tumor targeting, enhanced water solubility and biocompatibility, and PAMAM has good carrier gene guidance. The theoretical basis of cell entry is the breakthrough point. The HA-PAMAM/p EGFP gene transfer and expression system will be constructed to further study its physical and chemical properties, biological performance and targeting. The transfer system is optimized from PAMAM molecular weight, HA grafting rate and grafting density, polycation / gene weight ratio (w /w) and so on, and gene transfection efficiency is improved. The main contents of this study are: (1) the chemical synthesis and structural characterization of PAMAM-HA polymers: the use of hyaluronic acid (HA) and PAMAM in the buffer solution (P H8.5) of four sodium borate (Na BH4) containing Na Cl (P H8.5), and the formation of a PAMA-HA polymer under the catalysis of cyanoborohydride (Na BH3CN). The molecular weight, the HA grafting quantity and the graft density of PAMAM-HA samples were synchronized and compared with the.PAMAM-HA samples. The chemical structure of the.PAMAM-HA samples was identified as infrared (IR). (2) the performance of PAMAM-HA as a gene carrier: the particle size distribution and surface potential of the PAMAM-HA/DNA complex were measured by dynamic light scattering, and the composite was observed by transmission electron microscopy (TEM). Particle morphology; the ability of PAMAM-HA binding and compressing DNA by agarose gel block test. (3) PAMAM-HA cytotoxicity: MTT method was used to investigate the cytotoxicity of PAMAM-HA samples to different tumor cells and the toxicity of PAMAM-HA/DNA in the transfection ratio. (4) PAMAM-HA in vitro transfection study: Bel-7402 cells to PAMAM-HA/DNA The optimal transfection efficiency of the complex was screened and the transfection of PAMAM-HA/DNA complex between different cells was compared with PEI25K, and the effect of serum on transfection efficiency was investigated. (5) PAMAM-HA induced apoptosis: Annexin V-FITC cell apoptosis method was used to investigate whether various PAMAM-HA products could induce apoptosis at the experimental concentration. The relationship between apoptosis efficiency and concentration and time. (6) study on the mechanism of PAMAM-HA transfection: investigate the pathway of each compound into cells by different uptake inhibitors, and observe the distribution of DNA in cell by laser confocal microscopy, respectively, with the staining of nuclei, lysosomes and DNA.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450

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