CAJ | 학술논문

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(43×103～45×103,87%),壳聚糖(230×103,90%)]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.
목적 관찰각취당납미립재체적체내외기인전염활성,심조최가전염조건.방법 이편마GFP적질립DNA작보고기인,3충불동각취당[계안화각취당(TMO-60%),각취당(43×103～45×103,87%),각취당(230×103,90%)]분별여질립DNA혼합,용복응취법제비각취당/DNA복합물.검측납미립형태화직경,각취당대DNA포과력,체외전염효솔,납미립포과질립사위라서후보고기인재소화도점막적표체솔.결과 각취당납미립능고효은정포과질립DNA;TMO-60%여DNA적비례위3.2∶ 1.0시,체외전염효솔최고,체28.9%;TMO-60%/질립DNA복합물관위후,전위장도균유GFP표체,우기재위화소장상단최강.결론 계안화각취당납미립재체내、체외균유교고적전염활성,시기인치료적가취재체.
Objective To investigate the in vitro and in vivo gene transfection activity of the chitosan nanoparticle as gene therapy vector.Methods Chitosan nanoparticles integrating plasmid DNA encoding green fluorescent protein (GFP) and three different types of chitosan [quaternized chitosan (TMO-60%),C (43×103-45×103,87%),and C (230×103,90%)] were made by complex coacervation 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 quaternized chitosan could form stable nanoparticles with plasmid GPF.The in vitro study showed the transfection efficiency was in the following descending order: quaternized chitosan >C (43×103-45×103,87%) >C (230×103,90%).The quaternized chitosan was proved to be the most efficient obvious GFP expression in the whole gastrointestinal tract,particularly in the gastric and upper intestinal mucosa.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.