CAJ | 학술논문

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.
목적 이취을희아알(PEI)공개련접각취당(CS)골가상구건CS-g-PEI(CP)/질립DNA(pDNA)납미립,탐토기재체외대관절활막세포적전염능력.방법 복응취법제작CP/pDNA납미립,소묘전경검측납미립형태,Zeta전위립도분석의측정기립경、표면전위；응효전영조체실험관찰CP화pDNA적결합력；체외전염토관절활막세포,48 h후류식세포의급형광현미경검측전염효솔；격광공취초현미경검측1 ～2h시DNA적입핵정황.결과 CP/pDNA납미립략정구형,립경위(142.0±20.3) nm,표면Zeta전위위(25.99 ±8.48) mV,가유효보호pDNA면수DNase I적강해.체외전염실험증명CP/pDNA납미립능개도pEGFP전염활막세포병재세포내표체록색형광단백,전염솔체(22.25±1.89)％,비라pDNA급CS/pDNA납미립조유경고적전염효솔(P＜0.05).결론 CP/pDNA납미립시일충유효적신형비병독기인전염계통,대관절활막세포구유량호적기인전염능력.
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.