低分子量PEI接枝大豆蛋白基因载体的构建及转染性能研究

发布时间：2018-03-10 02:31

本文选题：基因治疗　切入点：大豆蛋白　出处：《安徽大学》2017年硕士论文　论文类型：学位论文

【摘要】：基因缺失和异常常导致许多疾病的发生,包括癌症,心血管疾病,遗传病等,这些疾病严重危害着人类的生命健康。基因治疗作为一种新型的生物治疗手段,因其能够安全、准确、有效的修正异变基因而备受关注。然而,如何设计适合的载体将目的基因高效、准确地运载到靶细胞中仍然是目前研究的重点与难点。聚乙烯亚胺(PEI)是普遍应用的阳离子聚合物基因载体之一,因其质子化之后具有高的电荷密度和良好的质子缓冲能力。其中,高枝化度后的PEI 25 KDa和22 KDa拥有高的转染效率,因而被称作聚阳离子基因载体领域中的"黄金准则"。但是高分子量PEI细胞毒性大,生物降解能力差,易与血清蛋白结合等严重制约了其作为基因载体的应用。另一方面,低分子量的PEI尽管细胞毒性弱,但是转染效率低,有些甚至几乎没有转染效率。为了解决低分子量PEI转染效率低的问题,近年来研究人员对低分子量PEI进行了多方面的研究。本论文基于天然大分子大豆分离蛋白为起始原料制备出一类简单、可生物降解、高效的的聚阳离子基因载体。首先对大豆分离蛋白进行酶解,接着离心和透析纯化,得到具有良好水溶性的低分子量大豆蛋白(SP)。将不同低分子量的PEI(600 Da,1200 Da,1800 Da)通过酰胺化反应修饰在大豆蛋白上得到一系列大豆蛋白-PEI复合产物(SP-PEIs),分别命名为SP-PEI600,SP-PEI1200,SP-PEI1800。经过红外和Zeta电位等实验证实,不同低分子量PEI成功地接枝到大豆蛋白上。通过酸碱滴定实验证明聚合物SP-PEIs的质子缓冲能力,通过蛋白吸附实验检测聚合物SP-PEIs对血清蛋白的抑制作用。接下来,复合物SP-PEIs/DNA的理化性质得到进一步详细研究。聚合物与DNA形成的复合物具有理想的粒径和电位有利于其快速进入细胞和提高基因转染。在pH为7.4的生理条件下,SP-PEIs与DNA形成的复合物随着N/P质量比的增加,复合物的粒径逐渐发生改变,最终会稳定在100-200nm左右,复合物SP-PEIs/DNA的电位也是随着N/P质量比的增大而增长。检测不同pH下复合物SP-PEIs/DNA粒径的变化,发现其在生理条件下(pH=7.4)可以稳定存在,在酸性条件下粒径发生明显变化,该特性有助于复合物上中的DNA从内涵体中逃逸出来。另外,通过琼脂糖电泳实验检测聚合物SP-PEIs对DNA的压缩能力,DNase I降解实验和肝素置换实验则进一步检测聚合物对DNA的压缩能力。之后,通过一系列细胞实验进一步评估了聚合物SP-PEIs作为基因载体的潜力和优势。枝化的PEI 25 KDa作为对照组,体外细胞毒性实验表明,在人肾上皮细胞(293T)、人神经母细胞瘤细胞(SH-SY5Y)共培养中,聚合物SP-PEIs均表现出比对照组PEI 25 KDa更低的毒性及良好的生物相容性。利用流式细胞仪和荧光倒置显微镜检测SP-PEIs/DNA复合物在有血清和无血清环境下的转染效率。结果证明,无论在有血清和无血清条件下复合物SP-PEIs/DNA在SH-SY5Y细胞系均有较高的转染效率。特别是聚合物SP-PEI1800与DNA在质量比为24时,无血清条件下转染效率(5.27%)是对照组(1.94%)的2.7倍,在有血清条件下则高达3.4倍;而在293T细胞中,无血清条件下聚合物SP-PEI1800与质粒DNA质量比为24的时候最高转染效率(15.1%)和对照组的转染效率(16.6%)几乎相当;同样的在有血清状态下,聚合物SP-PEI1800(8.19%)也能取得和对照组类似的转染效率(7.22%)。本论文制备的复合物SP-PEIs作为新型的基因载体,具有较低的细胞毒性,良好的生物相容性,同时能够降低血清蛋白的吸附,提高DNA在细胞中的的稳定性,并促进基因转染效率的提升。因此,在基因治疗的应用中,聚合物SP-PEIs作为基因载体拥有巨大的潜在应用价值。
[Abstract]:Gene deletion and abnormality often cause many diseases, including cancer, cardiovascular disease, genetic diseases, these diseases seriously endanger human health. Gene therapy is a new biological treatment, because it can be safe, accurate and effective correction of abnormal gene and concern. However, how to design suitable the carrier of the gene efficiently and accurately carried to the target cell is still the focus and difficulty in the present study. Polyethyleneimine (PEI) is one of the commonly used cationic polymer gene carrier, due to the protonation of with high charge density and good proton buffering capacity. Among them, the high branched PEI after 25 KDa and 22 KDa has high transfection efficiency, which is called the polycation gene carrier in the field of "gold standard". But the molecular weight of PEI cell toxicity, biodegradation ability is poor, and the serum Protein binding restrict its application as a gene carrier. On the other hand, the low molecular weight PEI despite weak cytotoxicity, but the transfection efficiency is low, some even almost no transfection efficiency. In order to solve the problem of low molecular weight PEI low transfection efficiency, in recent years, researchers of the low molecular weight of PEI the raw materials for the preparation of this paper. A kind of simple natural macromolecules based on soy protein isolate, biodegradable, high efficient cationic gene vector. Firstly, isolated soybean protein was hydrolyzed, then purified by centrifugation and dialysis, low molecular weight of soybean protein has good water solubility (SP). Different low molecular weight PEI (600 Da, 1200 Da, 1800 Da) through amidation reaction to obtain a series of modified soybean protein products in soybean protein composite -PEI (SP-PEIs), named as SP-PEI600, SP-PEI1200, SP-PEI1 800. by IR and Zeta potential experiments confirmed that different low molecular weight PEI was successfully grafted onto the soybean protein. The proton buffering capacity of acid-base titration experiments show that polymer SP-PEIs, the protein adsorption inhibition of polymer SP-PEIs assay on serum protein. Next, further detailed study of the physicochemical properties of SP-PEIs/DNA composites. The formation of complex polymer and DNA has ideal particle size and zeta potential for its rapid entry into the cell and enhance the gene transfection. At pH 7.4 under physiological conditions, the formation of complex SP-PEIs and DNA increased with the increase of N/ P mass ratio, composite particle size gradually changed, will eventually stabilize at 100-200nm about the potential of compound SP-PEIs/DNA is increased with increase of N/P mass ratio and detection. Different pH SP-PEIs/DNA complex changes in particle size, found under physiological conditions (pH= 7.4) can be stable under acidic conditions, particle size change, contribute to the complex on the properties of DNA endosomal escape. In addition, the polymer SP-PEIs agarose gel electrophoresis detection experiments on DNA compression capability, DNase I degradation and heparin replacement experiment is further detected on compression capability of polymer DNA. After that, through a series of experiments to further evaluate the cell polymer SP-PEIs as gene vector potential and advantages. The branched PEI 25 KDa as control group, showed that the in vitro cytotoxicity in human renal epithelial cells (293T), human neuroblastoma cells (SH-SY5Y) cultured in polymer SP-PEIs showed compared with the control group PEI 25 KDa low toxicity and good biocompatibility. By using flow cytometry and fluorescence microscope to detect SP-PEIs/DNA complexes in serum and serum-free environment Transfection efficiency. The results show that both in serum and serum free conditions of transfection efficiency under complex SP-PEIs/DNA in SH-SY5Y cell line was higher. Especially the polymer SP-PEI1800 and DNA in the ratio was 24, the transfection efficiency under serum-free conditions (5.27%) and control group (1.94%) is 2.7 times that in serum it is up to 3.4 times; and in 293T cells in serum-free conditions of polymer SP-PEI1800 and plasmid DNA mass ratio is 24 when the highest transfection efficiency (15.1%) and the control group transfection efficiency (16.6%) is almost the same in serum; under the condition of polymer SP-PEI1800 (8.19%) and the control can be achieved group similar transfection efficiency (7.22%). The preparation of compound SP-PEIs as a gene vector model, have low cytotoxicity and good biocompatibility, and can reduce the adsorption of serum proteins in the cell, improve DNA stability It is qualitative and promotes the enhancement of gene transfection efficiency. Therefore, in the application of gene therapy, polymer SP-PEIs has a great potential application value as a gene carrier.

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

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