新型纳米基因转移系统体外转染兔关节软骨细胞的研究  

A study of a novel nanometer gene transferring system and its transfection to chondrocytes in vitro in rabbits

作　　者：赵建宁[1] 王瑞[1] 郭亭[1] 高淑英[2] 张峻峰[2] 

机构地区：[1]南京军区南京总医院骨科研究所,210002 [2]南京大学生命科学院医药生物技术国家重点实验室

出　　处：《中华创伤骨科杂志》2007年第10期959-963,共5页Chinese Journal of Orthopaedic Trauma

基　　金：江苏省自然科学基金（BK2005085）;全军“十一五”科技攻关项目（06G043）

摘　　要：目的利用低相对分子质量壳聚糖制备一种新型载基因纳米微粒作为基因转移系统，表征其理化性质，检测其生物学特性，并评估其体外转染软骨细胞的性能。方法以增强型绿色荧光蛋白（EGFP）质粒或β-半乳糖酐酶质粒作为报告基因，通过分子自组装的方法制备载基因纳米壳聚糖微粒。以透射电镜观察微粒的形态，以激光粒径分析仪测定其粒径和表面的Zeta电位，行DNaseⅠ酶切保护实验研究壳聚糖对质粒DNA的保护作用，行噻唑兰试验研究纳米微粒对细胞的毒性作用，于体外行转染试验观察其对软骨细胞的转染活性。结果壳聚糖可以和目的基因质粒相互作用形成带有表面正电荷的纳米微粒，且能在很大程度上保护质粒DNA不受DNaseⅠ的降解，对细胞毒性小，转染软骨细胞后基因能够有效表达。结论载基因低相对分子质量壳聚糖纳米微粒能够有效地转染软骨细胞，是一种很有潜力的纳米局部传输系统。Objective To study the preparation and the characteristics of gene-loaded low molecular weight chitosan nanoparticles and their activity of transfection to chondrocytes in vitro in rabbits. Methods Gene-loaded low molecular weight chitosan nanoparticles were prepared by molecular self-assembly of low molecular weight chitosan and the reporter gene, enhanced green fluorescent protein （EGFP） or β-Galactosidase plasmid. The appearance of the nanoparticles was observed by transmission electron microscope（TEM）. The sizes and zeta potentials of the nanoparticles were measured by the laser particle analyzer. DNase Ⅰ assay was performed to study the gene potential of the nanoparticles. The cytotoxity of the nanoparticles was examined by the MTT assay. The transfection activity to the chondrocytes of the nanoparticles was evaluated by an in vitro gene transfection test. Results Nanoparticles with positive charges could be formed by the interaction of chitosan and plasmid, which protected the DNA from degradation by DNase Ⅰ to a large degree. The cytotoxity of the nanoparticles was insignificant. Genes could be effectively expressed in the chondrocytes, when the latter were transfected by the gene-loaded low molecular weight chitosan nanoparticles. Conclusion As gene-loaded low molecular weight chitosan nanoparticles can transfect chondrocytes effectively as vectors, they can function as a potential nanometer local delivery system.

关 键 词：壳多糖 纳米技术 转染 软骨细胞 

分 类 号：R686[医药卫生—骨科学]