摘要目的 制成以羟丁基壳聚糖(HBC)为壳,以巯基烷基化壳聚糖(TACS)包埋基因质粒为核的核壳结构,探讨其作为缓释基因载体的可行性.方法 TACS与增强型绿色荧光蛋白基因(pEGFP)以复凝聚法制成纳米微球作为核结构(TACS/pEGFP),然后与温敏性HBC混合包裹以及冷冻干燥合成羟丁基壳聚糖-巯基烷基化壳聚糖/pEGFP(HBC-TACS/pEGFP)核壳结构;利用动态光散射仪器、透射电镜对核及核壳结构进行表征.凝胶电泳实验检测复合基因纳米粒子对pEGFP的携载以及保护作用.对HEK293T细胞和小鼠的骨骼肌进行体外和体内转染,检测基因复合纳米粒能否作为缓释基因载体.结果 HBC-TACS/pEGFP纳米微球多呈球形,粒径为(317.6±55.9) nm,表面Zeta电位为(2.3 ±1.1) mV,可有效保护pEGFP免受DNase I的降解.在体外转染经流式细胞仪测定,HBC-TACS/pEGFP的基因转染率为(8.00±5.69)％,明显小于TACS/pEGFP[(37.66±5.18)％],差异有统计学意义(P＜0.01).但是在小鼠体内随着时间的推移,核壳结构组EGFP荧光蛋白表达量逐渐增加,第45天激光共聚焦显微镜下观察到HBC-TACS/pEGFP的荧光强度强于TACS/pEGFP.结论 HBC-TACS/pEGFP可以作为基因载体并且在体内有缓释基因,延长基因表达的作用.

abstractsObjective To prepare and evaluate the core-shell nanoparticles hydroxybutyl chitosanthiolated N-alkylated chitosan encapsulated with enhanced green fluorescent protein encoded plasmids (pEGFP) as a sustained-release gene carrier.Methods The pEGFP-loaded thiolated N-alkylated chitosan (TACS/pEGFP) was prepared by complex coacervation method as the core,and the core was coated with the thermosensitive HBC through mixed wrap and freeze drying,then the core-shell nanoparticles (HBC-TACS/pEGFP) were prepared.The nanoparticles were characterized by dynamic light scattering (DLS)and transmission electron microscope (TEM).Gel agarose electrophoresis was done to test the protective effects of the nanoparticles on the pEGFP.The nanoparticles were transfect into HEK293 cells in vitro and mouse skeletal muscle in vivo.Results HBC-TACS/pEGFP core-shell nanoparticles were mainly spherical,with an average size of (317.6 ±55.9) nm,and zeta potential of (2.3 ± 1.1) mV.The gel agarose electrophoresis confirmed that the nanoparticles could effectively protect the pEGFP from degradation against DNase I.In vitro gene transfection efficiency of HBC-TACS/pEGFP was (8.00 ± 5.69) ％,significantly lower than that of TACS/pEGFP [(37.66 ± 5.18)％],P ＜ 0.01.However,the expression of fluorescent protein was increased in the animal muscle in a time-dependent manner.On the day 45,the fluorescence intensity of HBC-TACS/pEGFP was higher than TACS/pEGFP.Conclusion HBC-TACS/pEGFP can be used as a gene vector which can be continuously expressed in vivo.