节点文献
壳聚糖纳米粒基因载体的制备及其转染研究
Preparation of Chitosan Nanoparticles as Gene Carriers and its Efficiency both in Vitro and in Vivo
【作者】 胡玉荣; 张强; 马萍; 吕万良; 王坚成; 张振中;
【Author】 HU Yurong 1,2 , ZHANG Qiang2, MA Ping3, LU Wanliang2, WANG Jiancheng2 , ZHANG Zhenzhong1 (1. School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China; 2. School of Pharmaceutical Sciences, Peking University, Beijing 100083, China; 3.The general Hospital of the second Artillery forces, Beijing 100088, China)
【机构】 大学药学院; 大学药学院药剂系; 第二炮兵总医院药剂科;
【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其在体外和体内实验中的转染能力。方法:用表达绿色荧光蛋白的质粒(pEGFP-C1)作报告基因,采用复凝聚法制备壳聚糖(chitosan,CS)/pEGFP-C1纳米粒(CS/pEGFP)。通过激光粒度测定仪和原子力显微镜对纳米粒的形态和粒径分布进行考察;应用凝胶阻滞和DNA酶消化实验检测CS对质粒基因(plasmidDNA,pDNA)的结合、保护作用;用体外和体内基因转染实验,评价纳米颗粒的转染能力;使用共聚焦显微镜对黑色素瘤(B16)细胞中所表达的GFP蛋白进行定位。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。制备的CS/pEGFP纳米粒为结构紧密的不规则球形。应用DNaseI消化后可见,CS可保护核酸免受破坏。CS/pEGFP纳米粒能成功转染B16细胞和人胚肾(HEK293)细胞及活体动物,GFP的绿色荧光在细胞质和细胞核中均表达。结论:壳聚糖可以有效凝聚pDNA,采用复凝聚法可制得200~400nm范围,荷正电的纳米粒。本研究制备的CS/pEGFP纳米粒在体外和体内均能实现有效转染,为基因治疗提供了一种潜在的载体。
【Abstract】 Purpose: To prepare chitosan (CS) nanoparticles (NPs)as gene carriers and study its pharmaceutical characteristics and gene transfection efficiency both in vitro and in vivo under optimizing the experimental condition. Methods: Nanoparticles of chitosan with plasmid DNA (pDNA, pEGFP-C1) encoding green fluorescenct protein (GFP) were prepared using a complex coacervation process and were characterized for their size, zeta potential, morphologies, their ability to bind and protect plasmid DNA from degradation. the confocal microscopy was used for the localization of the pEGFP protein in transfected Human melanoma(B16) cells. The transfection efficiency of CS/pEGFP NPs was tested both in vitro and in vivo. Results: The results of gel electrophoresis demonstrated full binding of chitosan with the pDNA by electrostatic interaction. The morphology of the chitosan NPs was mostly spherical and well distributed. CS can protect the pDNA From degradation by DNaseI. The green fluorescence of is evenly distributed in the cytosol and the nucleus in cells. As shown by fluorescen microscopy, green fluorescent protein reporter can be observed in the transfected cells(Human embryonic kidney (HEK 293) cells and B16 cells) as well as in the airway epithelium of the treated mice with CS/pEGFP NPs. Conclusions: Chitosan NP prepared by complex coacervation can bind to the pDNA efficiently and diameter distribution of 200 to 400 nm. These NPs allow efficient delivery of the reporter genes into cells in vitro and in vivo for their expression. The chitosan-pDNA NPs may serve as an effective non-viral carrier for delivery of nucleotides into cells.
- 【会议录名称】 第十届中国科协年会论文集(三)
- 【会议名称】第十届中国科协年会
- 【会议时间】2008-09
- 【会议地点】中国河南郑州
- 【分类号】TB383.1
- 【主办单位】中国科学技术协会、河南省人民政府