CAJ | 학술논문

目的以透明质酸(HA)修饰基因载体壳聚糖(CS)纳米微球,制作一种新型的HA/CS/pDNA基因转染系统,观察其结构特征及体外对软骨细胞的转染能力.方法将不同比例的HA和CS与负载增强型绿色荧光蛋白基因(EGFP)的质粒DNA(pDNA)以复凝聚法制成纳米微球,以扫描电镜检测纳米微球形态,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CS和pDNA的结合力;体外转染兔关节软骨细胞,以流式细胞仪及荧光显微镜检测转染效率.结果 HA/CS/pDNA纳米微球多呈球形,粒径为(223±51)nm,表面Zeta电位为(17.4±6.1)mV,可有效保护pDNA免受 DNaseⅠ的降解.体外转染实验证明HA/CS/pDNA纳米微球能介导pEGFP转染软骨细胞并在细胞内表达绿色荧光蛋白,转染率达(15.450±0.404)%,比裸pDNA组和CS/pDNA组有更高的转染效率(P<0.05).结论复凝聚法制备的HA/CS/pDNA纳米微球是一种有效的新型非病毒基因转染系统,对软骨细胞有着潜在的靶向基因转染能力.
목적 이투명질산(HA)수식기인재체각취당(CS)납미미구,제작일충신형적HA/CS/pDNA기인전염계통,관찰기결구특정급체외대연골세포적전염능력.방법 장불동비례적HA화CS여부재증강형록색형광단백기인(EGFP)적질립DNA(pDNA)이복응취법제성납미미구,이소묘전경검측납미미구형태,Zeta전위립도분석의측정기립경、표면전위;응효전영조체실험관찰CS화pDNA적결합력;체외전염토관절연골세포,이류식세포의급형광현미경검측전염효솔.결과 HA/CS/pDNA납미미구다정구형,립경위(223±51)nm,표면Zeta전위위(17.4±6.1)mV,가유효보호pDNA면수 DNaseⅠ적강해.체외전염실험증명HA/CS/pDNA납미미구능개도pEGFP전염연골세포병재세포내표체록색형광단백,전염솔체(15.450±0.404)%,비라pDNA조화CS/pDNA조유경고적전염효솔(P<0.05).결론 복응취법제비적HA/CS/pDNA납미미구시일충유효적신형비병독기인전염계통,대연골세포유착잠재적파향기인전염능력.
Objective To prepare hyaluronic acid (HA)-modified chitosan (CS)/pDNA (HA/CS/pDNA) nanoparticles as novel gene vectors, study their structural characteristics and gene transfection efficiency for chondrocytes in vitro in rabbits. Methods The HA/CS/pDNA nanoparticles were prepared by a DNA (pDNA), which load enhanced green fluorescent protein (EGFP) gene. The morphology of the nanoparticles was observed under the 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 with the chondrocytes of rabbits. The gene transfection efficiency was measured by using flow cytometry and under the fluorescence microscopy. Results HA/CS/pDNA nanoparticles were mainly spherical, with an average size of (223±51) nm, and zeta-potential of (17.4±6.1) mV. The agarose gel electrophoresis analysis confirmed that they could effectively protect pDNA from degradation against DNase I. Gene transfection in vitro proved that HA/CS/pDNA nanoparticles could be efficiently transfected into chondrocytes of rabbits, the expression of green fluorescent proteins was observed under the fluorescent microscopy, and the transfection efficiency reached as high as (15.450±0.404)%, significantly higher than that of the naked pDNA or the CS/pDNA nanoparticles (P<0.05). Conclusion HA/CS/pDNA nanoparticles were an effective novel non-viral gene transfer vector, which possessed the potential targeting transfection ability on chondrocytes.