【摘要】： 隨著納米生物技術的飛躍發(fā)展,以納米顆粒為基礎的非病毒載體系統(tǒng)的出現(xiàn),為實現(xiàn)安全高效的DNA轉(zhuǎn)染提供了更好的工具。其中無機納米顆粒載體通過表面恰當修飾可以克服機體各種屏障,靶向傳遞藥物或質(zhì)粒DNA,使其成為新型基因載體的研究熱點。硅是一種生物相容性好,低毒,表面易于修飾,適合用于納米基因載體的材料。目的:制備多聚賴氨酸-硅納米基因載體,并初步研究其轉(zhuǎn)染效果。方法: 第一部分選用微乳體系為壬基酚聚氧乙烯醚/正己醇/環(huán)己烷/雙蒸水,利用正硅酸乙酯在油包水型微乳體系中催化水解的方法制備硅納米。并用多聚賴氨酸為修飾物,使多聚賴氨酸通過靜電作用結(jié)合于硅納米表面,獲得基因載體-多聚賴氨酸-硅納米。 第二部分,通過瓊脂糖凝膠電泳,研究多聚賴氨酸-硅納米有效結(jié)合DNA的合適質(zhì)量比。 第三部分,將多聚賴氨酸-硅納米與綠色熒光蛋白質(zhì)粒(pEGFP -C1)結(jié)合在一起,體外轉(zhuǎn)染HepG2與L02,在倒置熒光顯微鏡下觀察轉(zhuǎn)染效率。 結(jié)果:制備了穩(wěn)定的油包水型微乳體系,并用該微乳液體系制備出大小均勻,粒徑30納米,穩(wěn)定性能好,分散性好的硅納米。以多聚賴氨酸修飾硅納米后,其與質(zhì)粒pEGFP-C1結(jié)合的合適質(zhì)量比為30:1。其轉(zhuǎn)染HepG2與L02的效率分別為35.1%與34.0%,與質(zhì)脂體轉(zhuǎn)染組效率相當(P5%)。 結(jié)論:制備了多聚賴氨酸-硅納米,該納米顆粒可作為基因載體,有效轉(zhuǎn)染質(zhì)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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R318.08;R346

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