负载pcDNA3.1-EGFP壳聚糖纳米颗粒的制备及其生物学特性分析  

Generation and characterization of a pcDNA3.1-EGFP plasmid delivery system based on chitosan-nanoparticles

作　　者：邵小青[1] 陈艳[1] 陈贝[1] 陈泽烨[1] 奚菊群[2] 龚卫娟[1] 

机构地区：[1]扬州大学医学院免疫学教研室,江苏扬州225001 [2]扬州大学医学院药剂学教研室,江苏扬州225001

出　　处：《实用临床医药杂志》2013年第17期1-4,共4页Journal of Clinical Medicine in Practice

基　　金：国家自然科学基金(81172785;30671917);江苏省自然科学基金(BK2011449;BK2008215);国家级大学生创新创业训练计划;江苏省大学生科技创新基金资助项目

摘　　要：目的 制备并鉴定负载pcDNA3.1-EGFP质粒的纳米颗粒,观察其细胞转染效率及细胞毒性.方法 首先将壳聚糖和质粒DNA按不同质量比混合,制备成纳米颗粒,计算其包封率,测定其粒径和Zeta电位.其次分别将纳米颗粒与RAW264.7和CT-26细胞孵育,荧光显微镜观察2两种细胞内绿色荧光蛋白的表达,流式细胞仪检测荧光细胞的频率.最后利用MTS/PMS法检测纳米颗粒对RAW264.7和CT-26细胞的毒性.结果 质粒DNA被壳聚糖纳米颗粒高效负载,粒径分布在100～300 nm之间,携带正电荷,被哺乳动物细胞有效摄取并表达,对细胞没有明显毒性.结论 负载DNA的壳聚糖纳米颗粒可用于重组基因疫苗的运送,可用于疾病的预防或治疗.Objective To generate and identify a pcDNA3.1-EGFP plasmid delivery system based on chitosan-nanoparticles, and to analyze cell-transfeeting efficiencies and cytotoxicities of these nanopartieles. Methods At first chitosan and plasmid DNA were mixed at a variety of mass ratios to form nanoparticles, and encapsulation efficiencies were calculated. Particle size and zeta potential were measured. Expression of green fluorescent protein in RAW264.7 and CT-26 cell lines was observed under microscopy after nanopartieels were incubated with above cells respectively. Frequencies of fluo- rescent cells were detected by flow cytometry. Finally cytotoxicities of nanopartieles on RAW264.7 and CT-26 cells were checked by a MTS/PMS assay. Results Plasmid DNA could be efficiently carried on chitosan-nanoparticles, and their sizes ranged from 100 nm to 300 nm and carried positive charges. These nanoparticles were uptaken and expressed efficiently by mammalian cells, and had no significant cytotoxeiyies against those cells. Conclusion Chitosan-nanopartricels as DNA-carriers can be used to deliver recombinant gene vaccines for prevention and therapy of diseases.

关 键 词：壳聚糖 纳米颗粒 DNA 

分 类 号：R392.33[医药卫生—免疫学]