多聚赖氨酸—硅纳米制备及体外转染细胞的实验研究
发布时间：2018-12-26 15:26
【摘要】： 随着纳米生物技术的飞跃发展,以纳米颗粒为基础的非病毒载体系统的出现,为实现安全高效的DNA转染提供了更好的工具。其中无机纳米颗粒载体通过表面恰当修饰可以克服机体各种屏障,靶向传递药物或质粒DNA,使其成为新型基因载体的研究热点。硅是一种生物相容性好,低毒,表面易于修饰,适合用于纳米基因载体的材料。目的:制备多聚赖氨酸-硅纳米基因载体,并初步研究其转染效果。方法: 第一部分选用微乳体系为壬基酚聚氧乙烯醚/正己醇/环己烷/双蒸水,利用正硅酸乙酯在油包水型微乳体系中催化水解的方法制备硅纳米。并用多聚赖氨酸为修饰物,使多聚赖氨酸通过静电作用结合于硅纳米表面,获得基因载体-多聚赖氨酸-硅纳米。 第二部分,通过琼脂糖凝胶电泳,研究多聚赖氨酸-硅纳米有效结合DNA的合适质量比。 第三部分,将多聚赖氨酸-硅纳米与绿色荧光蛋白质粒(pEGFP -C1)结合在一起,体外转染HepG2与L02,在倒置荧光显微镜下观察转染效率。 结果:制备了稳定的油包水型微乳体系,并用该微乳液体系制备出大小均匀,粒径30纳米,稳定性能好,分散性好的硅纳米。以多聚赖氨酸修饰硅纳米后,其与质粒pEGFP-C1结合的合适质量比为30:1。其转染HepG2与L02的效率分别为35.1%与34.0%,与质脂体转染组效率相当(P5%)。 结论:制备了多聚赖氨酸-硅纳米,该纳米颗粒可作为基因载体,有效转染质粒pEGFP -C1。
[Abstract]:With the rapid development of nanotechnology, the emergence of non-viral vector systems based on nanoparticles provides a better tool for the safe and efficient DNA transfection. Among them, inorganic nanoparticles can overcome all kinds of barriers by surface modification, and transfer drugs or plasmid DNA, to make them become the research focus of novel gene vectors. Silicon is a kind of materials with good biocompatibility, low toxicity and easy surface modification. Aim: to prepare poly-lysine-silicon nanogene vector and to study its transfection effect. Methods: in the first part, nonylphenol polyoxyethylene ether / n-hexanol / cyclohexane / double distilled water was used as microemulsion system to prepare silicon nanocrystalline by catalytic hydrolysis of ethyl orthosilicate in oil-in-water microemulsion system. Poly-lysine was used as modifier to bind poly-lysine to the surface of silicon nanocrystalline by electrostatic interaction, and the gene carrier, poly-lysine-silicon nanocrystalline, was obtained. In the second part, by agarose gel electrophoresis, the proper mass ratio of poly (lysine-silicon) nanobound DNA was studied. In the third part, poly-lysine-silicon nanoparticles were combined with green fluorescent protein particles (pEGFP C1) to transfect HepG2 and L02 in vitro, and the transfection efficiency was observed under inverted fluorescence microscope. Results: the stable oil-in-water microemulsion system was prepared and the size of the microemulsion was 30 nm with good stability and dispersity. After modified with polylysine, the suitable mass ratio of the modified silica nanoparticles to plasmid pEGFP-C1 was 30: 1. The efficiency of transfection of HepG2 and L02 was 35.1% and 34.0respectively, which was similar to that of lipids transfection group (P5%). Conclusion: Poly (Lysine-Si) nanoparticles were prepared. The nanoparticles can be used as gene vectors for transfection of plasmid pEGFP C1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R318.08;R346
本文编号：2392318
[Abstract]:With the rapid development of nanotechnology, the emergence of non-viral vector systems based on nanoparticles provides a better tool for the safe and efficient DNA transfection. Among them, inorganic nanoparticles can overcome all kinds of barriers by surface modification, and transfer drugs or plasmid DNA, to make them become the research focus of novel gene vectors. Silicon is a kind of materials with good biocompatibility, low toxicity and easy surface modification. Aim: to prepare poly-lysine-silicon nanogene vector and to study its transfection effect. Methods: in the first part, nonylphenol polyoxyethylene ether / n-hexanol / cyclohexane / double distilled water was used as microemulsion system to prepare silicon nanocrystalline by catalytic hydrolysis of ethyl orthosilicate in oil-in-water microemulsion system. Poly-lysine was used as modifier to bind poly-lysine to the surface of silicon nanocrystalline by electrostatic interaction, and the gene carrier, poly-lysine-silicon nanocrystalline, was obtained. In the second part, by agarose gel electrophoresis, the proper mass ratio of poly (lysine-silicon) nanobound DNA was studied. In the third part, poly-lysine-silicon nanoparticles were combined with green fluorescent protein particles (pEGFP C1) to transfect HepG2 and L02 in vitro, and the transfection efficiency was observed under inverted fluorescence microscope. Results: the stable oil-in-water microemulsion system was prepared and the size of the microemulsion was 30 nm with good stability and dispersity. After modified with polylysine, the suitable mass ratio of the modified silica nanoparticles to plasmid pEGFP-C1 was 30: 1. The efficiency of transfection of HepG2 and L02 was 35.1% and 34.0respectively, which was similar to that of lipids transfection group (P5%). Conclusion: Poly (Lysine-Si) nanoparticles were prepared. The nanoparticles can be used as gene vectors for transfection of plasmid pEGFP C1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R318.08;R346
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