壳聚糖纳米粒作为消化道给药基因治疗载体的研究  

Experimental studies on chitosan nanoParticle as gene therapy vector via gastrointestinal mucosa

作　　者：郑芳[1] 李雁[1] 施晓文[2] 杨桂芳[1] 龚玲玲[1] 袁宏银[1] 杜予民[2] 崔冶建[3] 汪艳[3] 

机构地区：[1]武汉大学中南医院肿瘤科肿瘤生物学行为湖北省重点实验室,430071 [2]武汉大学环境与资源学院 [3]武汉大学生物医学结构中心

出　　处：《中华实验外科杂志》2008年第6期767-769,共3页Chinese Journal of Experimental Surgery

基　　金：教育部新世纪优秀人才支持计划（NCET-04-0669）;全国优秀博士学位论文作者专项资金资助项目（200464）;湖北省青年杰出人才基金资助项目（301161202）

摘　　要：目的观察壳聚糖纳米粒载体的体内外基因转染活性，寻找最佳转染条件。方法以编码GFP的质粒DNA作报告基因，3种不同壳聚糖[季铵化壳聚糖（TMO-60％），壳聚糖（43×10^3～45×10^3，87％），壳聚糖（230×10^3，90％）]分别与质粒DNA混合，用复凝聚法制备壳聚糖／DNA复合物。检测纳米粒形态和直径，壳聚糖对DNA包裹力，体外转染效率，纳米粒包裹质粒饲喂裸鼠后报告基因在消化道黏膜的表达率。结果壳聚糖纳米粒能高效稳定包裹质粒DNA；TMO-60％与DNA的比例为3．2：1．0时，体外转染效率最高，达28．9％；TMO-60％／质粒DNA复合物灌胃后，全胃肠道均有GFP表达，尤其在胃和小肠上段最强。结论季铵化壳聚糖纳米粒在体内、体外均有较高的转染活性，是基因治疗的可取载体。Objective To investigate the in vitro and in vivo gene transfection activity of the chi- tosan nanoparticle as gene therapy vector. Methods Chitosan nanoparticles integrating plasmid DNA encoding green fluorescent protein （GFP） and three different types of chitosan [quatemized chitosan （ TMO- 60% ）.C （43 × 10^3-45 × 10^3, 87% ）, and C （230 × 10^3. 90% ）] were made by complex coacervatlon process. The shape and size of the particles were studied by transmission electron microscopy. The chitosan-DNA binding capability was detected by gel electrophoresis. The optimal chitosan： DNA ratio for maximal in vitro transfection was studied by adding various chitosan nanoparticles to the cell culture of human hepatocellular carcinoma cell line HCCLM6. The chitosan nanoparticles were fed to 12 BALB/C-nu/ nu nude mice via a gastric feeding tube,2 times a week for a month,by the end of which the in vivo transfection efficiency of the gastrointestinal tract mucosa was studied. Negative control and blank control were set up at the same time. Results Both conventional and quatemized chitosan could form stable nanoparticles with plasmid GPF. The in vitro study showed the transfection efficiency was in the following descending order： quatemized chitosan 〉 C （43 × 10^3-45 × 10^3 .87% ） 〉 C （230 × 10^3 ,90% ）. The quaternized chitosml was proved to be the most efficient and the optimal chitosan/DNA ratio being 3.2： 1. In vivo study showed obvious GFP expression in the whole gastrointestinal tract, particularly in the gastric and upper intestinal mueosa. Conclusion The quaternized chitosan nanoparticle has relatively high in vitro and in vivo transfection activity, with minimal toxicity, which made it a desirable non-viral vector for gene therapy.

关 键 词：壳聚糖 纳米粒 基因治疗 胃肠道 

分 类 号：R318.08[医药卫生—生物医学工程]