透明质酸／壳聚糖/pEGFP纳米粒介导体外基因转染关节软骨细胞与滑膜细胞的比较

目的]以透明质酸(HA)修饰的壳聚糖(CS)／质粒DNA(pDNA)纳米粒介导体外基因转染关节软骨细胞与滑膜细胞,以明确其作为非病毒基因载体治疗关节疾病的潜能.方法]将HA修饰的CS与负载增强型绿色荧光蛋白基因(EGFP)的pDNA以复凝聚法制成纳米粒,以扫描电镜检测纳米粒形态；激光粒度仪测定其粒径、Ze-ta电位及分散度(PDl)；凝胶电泳阻滞试验榆测HA/CS和pDNA的结合力及pDNA的释放；体外转染兔关节软骨细胞与滑膜细胞,以流式细胞仪及荧光显微镜检测转染效率.结果]HA/CS/pDNA纳米粒多呈球形,粒径平均为(142.5±20.3)nm,表面Zeta电位平均为(25.99±8.48)mV,分散度平均为(0.283±0.089),可有效保护pDNA免受核酸酶的降解；通过调节pH值至7.5以上或加入壳聚糖酶可促使纳米粒中的pDNA释放；体外转染实验证明HA/CS/pDNA纳米粒能介导pEGFP转染软骨细胞和滑膜细胞并在细胞内表达绿色荧光蛋白,其对软骨细胞的转染能力较强,比裸pEGFP和CS/pEGFP纳米粒有更高的转染效率(P＜0.05)；但对滑膜细胞的基因转染效率较低,与CS/pEGFP纳米粒无明显差别(P＞0.05).结论]复凝聚法制备的HA/CS/pDNA纳米粒是一种有效的新型非病毒基因转染载体,在体外可介导基因转染关节软骨细胞和滑膜细胞,其转染效率具有明显的细胞依赖性.

Hyaluronic acid/chitosan/pEGFP nanoparticles mediated gene transfection of articular chondrocytes and synoviocytes in vitro

Objective]Hyaluronic acid(HA) was used to modify chitosan/pDNA nanoparticles to produce HA/CS/pDNA nanoparticles as novel gene vectors.And to study HA/CS/pDNA nanoparticles mediated gene transfection of chondrocytes and synoviocytes in vitro,so as to ascertain them as potential non-viral gene vectors for the treatment of joint disease.Methods]The HA/CS/pDNA nanoparticles were prepared by a complex coacervation method with HA modified chitosan mixed with plasmid DNA(pDNA),which loaded enhanced green fluorescent protein(EGFP) gene.The morphology of nanoparticles was observed under scanning electron microscopy.The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer.The binding of pDNA with chitosan and the release of pDNA from HA/CS/pDNA nanoparticles were evaluated by agarose gel electrophoresis analysis.The gene transfection experiments in vitro were performed with rabbit’s chondrocytes and synoviocytes.The gene transfection efficiency was measured by flow cytometry and fluorescence microscope.Results]HA/CS/pDNA nanoparticles were mainly spherical with an average size of(142.5±20.3) nm,zeta-potential of(25.99±8.48) mV and polydispersity index of(0.283±0.089).The agarose gel electrophoresis analysis confirmed that they can effectively protect pDNA from degradation against DNase I.By adjusting the pH value to 7.5 or even higher,or adding chitosanase,the release of pDNA from the nanoparticles could be promoted.Gene transfection in vitro proved that HA/CS/pDNA nanoparticles were efficient in transfecting rabbit’s chondrocytes and synoviocytes,and the expressions of green fluorescent proteins were observed under fluorescent microscope.And their transfection efficiency against chondrocytes were much higher than that of synoviocytes,and also,were much higher than that of the naked pDNA or the CS/pDNA when transfected chondrocytes.Conclusion]HA/CS/pDNA nanoparticles were effective novel non-viral gene transfer vectors,which could mediate gene transfering of articular chondrocytes and synoviocytes.Although their transfection efficiency was cell dependent,they provided potential targeting non-viral gene transfer vectors for joint disease.