摘要
目的 以聚乙烯亚胺（PEI）共价连接壳聚糖（CS）骨架上构建CS-g-PEI（CP）/质粒DNA（pDNA）纳米粒,探讨其在体外对关节滑膜细胞的转染能力.方法 复凝聚法制作CP/pDNA纳米粒,扫描电镜检测纳米粒形态,Zeta电位粒度分析仪测定其粒径、表面电位；凝胶电泳阻滞实验观察CP和pDNA的结合力；体外转染兔关节滑膜细胞,48 h后流式细胞仪及荧光显微镜检测转染效率；激光共聚焦显微镜检测1 ～2h时DNA的入核情况.结果 CP/pDNA纳米粒略呈球形,粒径为（142.0±20.3） nm,表面Zeta电位为（25.99 ±8.48） mV,可有效保护pDNA免受DNase I的降解.体外转染实验证明CP/pDNA纳米粒能介导pEGFP转染滑膜细胞并在细胞内表达绿色荧光蛋白,转染率达（22.25±1.89）％,比裸pDNA及CS/pDNA纳米粒组有更高的转染效率（P＜0.05）.结论 CP/pDNA纳米粒是一种有效的新型非病毒基因转染系统,对关节滑膜细胞具有良好的基因转染能力.
Objective Polyethylenimine （PEI） was covalcntly linked to chitosan to construct CS-g-PEI （CP）/DNA nanoparticles as a noval non-viral gene transfection system,then to investigate its transfection efficiency in synoviocytes in vitro.Methods The CP/plasmid DNA （pDNA） nanoparticles were prepared by a complex coacervation method with synthesized CP mixed with pDNA,which loaded enhanced green fluorescent protein （EGFP） gene.The nanoparticles＇ morphology was observed under scanning transmission electron microscopy.The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer.The binding of pDNA was evaluated by agarose gel electrophoresis analysis.The gene transfection experiments in vitro were performed on rabbit＇ s synoviocytes.The gene transfection efficiency was measured by using flow cytometry and under fluorescence microscope at 48 h post-incubation.The entry of marked DNA into the nucleus of synoviocytes mediated by CP/pDNA was detected by using laser scanning confocal microscopy in 1-2 h.Results CP/pDNA nanoparticles were mainly spherical,with an average size of （142.0 ± 20.3） nm,and zeta-potential of （25.99 ± 8.48） mV.The agarose gel electrophoresis analysis confirmed that CP/pDNA nanoparticles could effectively protect pDNA from degradation against DNase I.Gene transfection in vitro proved that CP/pDNA was efficient in transfecting rabbit＇ s synoviocytes and the expression of green fluorescent proteins was observed under fluorescent microscope,and its transfection efficiency reached as high as （22.25 ± 1.89） %,which was significantly higher than that of the naked pDNA.Conclusion CP/pDNA nanoparticles were an effective novel non-viral gene vector,which possessed the potential favourable transfection ability towards synoviocytes.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2358-2360,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81272040）
广东省自然科学基金资助项目（S2011010004808）
关键词
壳聚糖
聚乙烯亚胺
基因载体
滑膜细胞
Keywords：Chitosan
Polyethylenimine
Gene vector
Synoviocyte
