聚乙烯亚胺修饰纳米金基因载体制备及体外实验研究

发布时间：2018-01-17 03:14

  本文关键词：聚乙烯亚胺修饰纳米金基因载体制备及体外实验研究　出处：《第三军医大学学报》2016年09期 　论文类型：期刊论文

  更多相关文章： 聚乙烯亚胺 纳米金 基因载体 转染

【摘要】：目的制备聚乙烯亚胺修饰纳米金基因载体并研究其理化性质的表征参数和体外转染效率。方法通过化学还原法制备聚乙烯亚胺修饰的纳米金基因载体,用绿色荧光蛋白质粒(pAc GFPN1)做报告基因,纳米基因载体可通过静电吸附的方式结合质粒DNA。用紫外分光光度计检测其吸收光谱,用透射电镜观察其形态特征,激光粒度分析仪测定其粒度分布、表面电位(Zeta电位),1%琼脂糖凝胶电泳检测该基因载体与质粒DNA的结合稳定性,CCK-8实验检测聚乙烯亚胺修饰纳米金基因载体及DNA-纳米金复合物对HEK293细胞的细胞毒性作用,通过荧光显微镜观察聚乙烯亚胺纳米基因载体介导pAcGFP-N1在体外培养的HEK293细胞中的表达,并分析其转染效率。结果聚乙烯亚胺还原氯金酸可以得到带正电荷的纳米颗粒,呈单分散球形分布,其粒径为(12.3±3.3)nm。在pH=7.2时,Zeta电位为+(29.7±5.1)m V。1%琼脂糖凝胶电泳结果表明,当纳米金/质粒DNA≥0.5时,质粒DNA可完全结合到纳米金表面。体外转染实验表明,聚乙烯亚胺修饰纳米金基因载体能介导pAcGFPN1转染HEK293细胞并在细胞中表达绿色荧光蛋白,其转染效率可达25%。结论聚乙烯亚胺修饰纳米金是一种新型非病毒基因载体,具有转染效率高、对细胞毒性小等优势。
[Abstract]:Objective to prepare polyvinyleneimine modified nano-gold gene vector and study its physical and chemical properties and transfection efficiency in vitro. Methods Polyvinyleneimine modified nano-gold gene vector was prepared by chemical reduction method. Using the green fluorescent protein particle pAc GFPN1 as the reporter gene, the nano-gene vector could bind to the plasmid DNA by electrostatic adsorption. The absorption spectrum of the vector was detected by UV spectrophotometer. The morphology of the plasmid was observed by transmission electron microscope, the particle size distribution was measured by laser particle size analyzer, and the binding stability of the gene vector to plasmid DNA was detected by 1% agarose gel electrophoresis. The cytotoxic effects of polyimide modified gold gene carrier and DNA-nano-gold complex on HEK293 cells were detected by CCK-8 assay. The expression of pAcGFP-N1 in HEK293 cells was observed by fluorescence microscope. The transfection efficiency was analyzed. Results the positively charged nanoparticles could be obtained by polyimide reduction with chloruronic acid, and the particle size was 12. 3 卤3. 3nm. at pH=7.2. The Zeta potential was 29.7 卤5.1mV.1% agarose gel electrophoresis. The results showed that the gold nanoparticles / plasmid DNA 鈮，

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