中文摘要：

目的制备载基因壳聚糖纳米粒,研究纳米粒的结构特征以及对细胞的基因转染效率。方法用表达绿色荧光蛋白的质粒（pGFP）作报告基因,采用复凝聚法制备壳聚糖-pGFP纳米粒。琼脂糖凝胶电泳分析壳聚糖和pDNA的结合能力,通过比色法检测其包封率,用纳米粒度仪和原子力显微镜对纳米粒的形态和粒径分布进行考察;通过荧光显微镜观察壳聚糖纳米粒介导pGFP在体外培养的人结肠腺癌细胞LoVo中的表达。结果琼脂糖凝胶电泳分析结果表明,pDNA与壳聚糖之间通过静电作用而完全结合,包封率大于90%。制备的壳聚糖-pGFP纳米粒为结构紧密的不规则球形,平均粒径为209nm,多分散指数为0.15。体外细胞转染的结果表明,壳聚糖-pGFP纳米粒能介导pGFP转染LoVo细胞并在细胞中表达绿色荧光蛋白。结论壳聚糖可以有效凝聚pDNA,采用复凝聚法可制得100￣500nm范围荷正电的纳米粒,有较高的包封率。壳聚糖纳米粒在体外能将基因递送到细胞内,并且报告基因能在细胞内表达。因此,壳聚糖作为非病毒基因载体具有介导核酸类生物大分子的应用价值。

英文摘要：

Objective To prepare chitosan nanoparticles （NPs） as gene carriers and study its pharmaceutical characteristics and gene transfection efficiency in vitro, Methods The plasmid expressing green fluorescent protein （pGFP） was used as the reporter gene, and the chitosan-pGFP NPs were prepared using a complex coacervation process. The binding ofpDNA was evaluated by agarose gel electrophoresis analysis, and the encapsulation rate was determined with colorimetry. The size distribution and polydispersity of the NPs were measured by nanoparticle size analyzer, and their morphologies observed by atomic force microscope. The transfection studies were performed with LoVo cells in vitro. Results The results of gel electrophoresis demonstrated full binding ofchitosan with the pDNA by electrostatic interaction. The encapsulation rates for these NPs all exceeded 90%. The morphology of the chitosan NPs was mostly spherical and well distributed, with a mean diameter of about 209 nm and polydispersity of 0.32. The in vitro transfection of chitosan-pGFP NP was efficient in LoVo cells and the expression of green fluorescent proteins was observed under fluorescent microscope. Conclusions Chitosan NP prepared by complex coacervation can bind to the pDNA efficiently with high encapsulation rate and diameter distribution of 100 to 500 nm. These NPs allow efficient delivery of the reporter genes into cells in vitro for their expression. The chitosan-pDNA NPs may serve as an effective non-viral carrier for delivery ofnucleotides into cells.