基于聚乙烯亚胺包裹的纳米金颗粒对PCR的优化研究及其在基因传递中的研究
发布时间：2018-09-06 14:23
【摘要】：随着纳米科技的快速发展,采用纳米技术制备的纳米材料成为了当前的研究热点。纳米材料由于具有特殊的物理化学性质,已经被广泛应用于生物医学领域,并取得了很多有价值的应用成果。聚合酶链式反应(Polymerase Chain Reaction,PCR技术)是一种基于DNA扩增的分子生物学技术,也是临床医学中的检测疾病的重要手段,但是PCR扩增中时常出现的低效率和非特异性问题限制了该技术的广泛应用。“纳米PCR”概念的引进为PCR技术的完善提供了一种独特的思路。此外,基因治疗(Gene therapy)作为一种新型的治疗疾病的方法被认为是药学和医学领域的一次革命。与传统治疗方法相比,基因治疗有望从根本上治疗疾病,已成为目前国际学术界研究的焦点。然而,基因传递中使用的基因传递载体经常具有很高的毒性或者较低的转染效率,以纳米材料为代表的非病毒载体逐渐成为科学家们研究的“新宠”。本硕士论文主要是以超支化聚乙烯亚胺(Polyethylenimine,PEI)包裹的纳米金颗粒作为优化剂应用于PCR技术,并探讨其优化机理,同时作为一种负载外源正常基因的传递载体应用于基因治疗的研究。我们希望通过本论文的研究,开发出一种既可以优化PCR技术又能作为理想的基因传递载体的多功能性的纳米材料。在第二章中,首先我们通过选择价廉易得的PEI作为树状大分子的替代品,在PEI表面修饰聚乙二醇(mPEG),用来提高纳米材料的稳定性和生物相容性。并以此为模板,利用硼氢化钠还原法,调控氯金酸与PEI的投料摩尔比,从而调控纳米材料的结构,表面电势和粒径大小,最终合成了一系列包裹不同数量纳米金颗粒的PEG-Au PENPs(PEGylated PEI-entrapped Entrapped Gold Nanoparticles)。另外,通过表征数据可知:所合成的PEG-Au PENPs粒径较小、分布均匀、具有良好的生物相容性和较高的热导率。在第三章中,根据相关文献,我们首先建立了两种PCR扩增体系:二轮易错PCR反应体系和高GC含量(74%GC含量)PCR反应体系。然后以这两种体系作为PCR优化剂的筛选平台,通过一系列优化实验和凝胶电泳实验来评估PEG-Au PENPs的优化能力。此外,我们通过系统的实验设计,进一步探讨了纳米材料对PCR技术的优化机理。结果表明:所有的PEG-Au PENPs都可以提高PCR反应的效率和特异性。值得指出的是,我们发现纳米材料在不同的PCR体系中,可能展示着不同的优化机理。在二轮易错PCR体系中,PEG-Au PENPs的优化作用可能是由于其表面电荷介导的静电作用所导致的;而在高GC含量PCR体系中,PEG-Au PENPs的良好的热传递性能可能是优化PCR反应的主要原因。在第四章中,我们首先通过凝胶阻滞实验测试了PEG-Au PENPs压缩pDNA的能力,然后通过增强型绿色荧光蛋白(EGFP)基因和荧光素酶(Luc)基因的表达实验来衡量材料的转染效率。最后,通过流式细胞术以及材料/pDNA复合物在细胞内的定位实验进一步研究了材料的转染机理。结合第二章的细胞毒性实验,我们发现:与PEI相比,PEG-Au PENPs不仅可以明显的降低细胞毒性,而且能保持较高的转染效率,另外,PEG-Au PENPs/pDNA复合物与PEI/pDNA复合物在细胞内有着共同的胞内传递途径。
[Abstract]:With the rapid development of nanotechnology, nanomaterials prepared by nanotechnology have become the focus of current research. Nanomaterials have been widely used in biomedical fields due to their special physical and chemical properties. Many valuable applications have been achieved. Polymerase Chain Reaction (PCR) Technology The introduction of the concept of "nano-PCR" provides a unique way of thinking for the improvement of PCR technology. In addition, gene therapy Gene therapy, as a new method of treating diseases, is regarded as a revolution in pharmacy and medicine. Compared with traditional therapy, gene therapy is expected to treat diseases fundamentally, and has become the focus of international academic research. However, gene delivery vectors used in gene delivery are often highly toxic. Non-viral vectors, such as nano-materials, have gradually become the "new favorite" of scientists because of their low transfection efficiency.In this dissertation, gold nanoparticles encapsulated by Hyperbranched polyethylenimine (PEI) were used as optimizers in PCR, and their optimization mechanism was discussed as well as as as a load. We hope to develop a kind of multifunctional nano-material which can optimize PCR technology and can be used as an ideal gene delivery vector. In the second chapter, we first choose the cheap and easy-to-obtain PEI as a substitute for dendrimers. Polyethylene glycol (mPEG) was modified on the surface of PEI to improve the stability and biocompatibility of nano-materials. As a template, sodium borohydride reduction method was used to adjust the molar ratio of chloroauric acid to PEI, so as to control the structure, surface potential and particle size of nano-materials. Finally, a series of nano-gold particles were synthesized. PEG-Au PENPs (PEGylated PEI-entrapped Entrapped Gold Nanoparticles). In addition, the characterization data showed that the synthesized PEG-Au PENPs had small particle size, uniform distribution, good biocompatibility and high thermal conductivity. R reaction system and high GC content (74% GC) PCR reaction system were used as the screening platform for PCR optimizers. A series of optimization experiments and gel electrophoresis experiments were carried out to evaluate the optimization ability of PEG-Au PENPs. The results showed that all PEG-Au PENPs could improve the efficiency and specificity of PCR reaction. It is worth pointing out that nanomaterials may exhibit different optimization mechanisms in different PCR systems. In Chapter 4, we first tested the ability of PEG-Au PENPs to compress pDNA by gel blockade, and then confirmed the expression of enhanced green fluorescent protein (EGFP) and luciferase (Luc) genes. Finally, the transfection mechanism of PEG-Au PENPs was further studied by flow cytometry and the localization of material/pDNA complex in cells. Combined with the cytotoxicity experiment in Chapter 2, we found that PEG-Au PENPs not only significantly reduced the cytotoxicity of PEI, but also maintained a high level of transfection. In addition, PEG-Au PENPs/pDNA complex and PEI/pDNA complex share the same intracellular transmission pathway.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450
[Abstract]:With the rapid development of nanotechnology, nanomaterials prepared by nanotechnology have become the focus of current research. Nanomaterials have been widely used in biomedical fields due to their special physical and chemical properties. Many valuable applications have been achieved. Polymerase Chain Reaction (PCR) Technology The introduction of the concept of "nano-PCR" provides a unique way of thinking for the improvement of PCR technology. In addition, gene therapy Gene therapy, as a new method of treating diseases, is regarded as a revolution in pharmacy and medicine. Compared with traditional therapy, gene therapy is expected to treat diseases fundamentally, and has become the focus of international academic research. However, gene delivery vectors used in gene delivery are often highly toxic. Non-viral vectors, such as nano-materials, have gradually become the "new favorite" of scientists because of their low transfection efficiency.In this dissertation, gold nanoparticles encapsulated by Hyperbranched polyethylenimine (PEI) were used as optimizers in PCR, and their optimization mechanism was discussed as well as as as a load. We hope to develop a kind of multifunctional nano-material which can optimize PCR technology and can be used as an ideal gene delivery vector. In the second chapter, we first choose the cheap and easy-to-obtain PEI as a substitute for dendrimers. Polyethylene glycol (mPEG) was modified on the surface of PEI to improve the stability and biocompatibility of nano-materials. As a template, sodium borohydride reduction method was used to adjust the molar ratio of chloroauric acid to PEI, so as to control the structure, surface potential and particle size of nano-materials. Finally, a series of nano-gold particles were synthesized. PEG-Au PENPs (PEGylated PEI-entrapped Entrapped Gold Nanoparticles). In addition, the characterization data showed that the synthesized PEG-Au PENPs had small particle size, uniform distribution, good biocompatibility and high thermal conductivity. R reaction system and high GC content (74% GC) PCR reaction system were used as the screening platform for PCR optimizers. A series of optimization experiments and gel electrophoresis experiments were carried out to evaluate the optimization ability of PEG-Au PENPs. The results showed that all PEG-Au PENPs could improve the efficiency and specificity of PCR reaction. It is worth pointing out that nanomaterials may exhibit different optimization mechanisms in different PCR systems. In Chapter 4, we first tested the ability of PEG-Au PENPs to compress pDNA by gel blockade, and then confirmed the expression of enhanced green fluorescent protein (EGFP) and luciferase (Luc) genes. Finally, the transfection mechanism of PEG-Au PENPs was further studied by flow cytometry and the localization of material/pDNA complex in cells. Combined with the cytotoxicity experiment in Chapter 2, we found that PEG-Au PENPs not only significantly reduced the cytotoxicity of PEI, but also maintained a high level of transfection. In addition, PEG-Au PENPs/pDNA complex and PEI/pDNA complex share the same intracellular transmission pathway.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450
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