壳聚糖-负载增强型绿色荧光蛋白基因的质粒DNA纳米微球体外转染软骨细胞的能力及影响因素

目的 探讨壳聚糖介导体外基因转染软骨细胞的能力及不同条件下基因转染率的变化,以筛选最佳转染条件.方法 将壳聚糖与负载增强型绿色荧光蛋白(EGFP)基因的质粒DNA(pDNA)以复凝聚法制成壳聚糖/pEGFP纳米微球,用扫描电镜检测纳米微球的形态,Zeta电位粒度分析仪测定其粒径、表面电位及分散度.以脂质体为对照,观察对软骨细胞的毒性.体外转染兔关节软骨细胞,以裸pDNA及脂质体为对照,流式细胞仪及荧光显微镜检测基因转染率.检测在不同pH值、N/P比值及pDNA剂量下壳聚糖/pEGFP纳米微球介导对软骨细胞的转染率变化.结果 壳聚糖/pEGFP纳米微球呈球形,平均粒径为(141.5±26.7)nm,表面Zeta电位平均为(17.8±3.9)mV,分散度平均为0.227±0.025.细胞毒性试验显示壳聚糖/pEGFP纳米微球与软骨细胞相容性良好,与脂质体比较差异有统计学意义(P＜0.05).体外转染实验证实壳聚糖/pEGFP纳米微球能转染软骨细胞并在细胞内表达绿色荧光蛋白,在pH值为7.0、N/P为5、pDNA浓度为4.0μg/mL时基因转染率最高,48 h转染率达10.9％±0.2％.结论 复凝聚法制备的壳聚糖/pEGFP纳米微球是一种有效的非病毒基因转染系统,细胞毒性小,对软骨细胞有一定基因转染能力,其转染率与pH值、N/P比值及pDNA剂量等密切相关,pH值为7.0、N/P为5、pDNA浓度为4.0 μg/mL是其最佳转染条件.

Transfection of chondrocytes with chitosan-pDNA loaded with enhanced green fluorescent protein nanoparticles in vitro and its influencing factors

Objective To investigate the optimal conditions for transfection of chondrocytes with chitosan-pEGFP nanoparticles in vitro in rabbits.Methods By a complex coacervation method,chitosan was mixed with plasmid DNA(pDNA)loaded with enhanced green fluorescent protein(EGFP)gene to prepare chitosan-pEGFP nanoparticles.Morphological features of the nanoparticles were observed under scanning electron microscopy.Sizes,zeta-potentials and polydispersity indexes of the nanoparticles were measured by a Marven-nano laser diffractometer.Using LipofectamineTM 2000 as a control,the cytotoxicity was examined against chondrocytes.Also,with naked pEGFP and LipofectamineTM 2000 as control,the gene transfection experiments in vitro were performed against chondrocytes.The gene transfection efficiency was measured with flow cytometry and under fluorescence microscopy.At the same time,variations in transfection efficiency of chitosan-pEGFP nanoparticles were tested at different pH values,N/P ratios and plasmid DNA doses.Results Chitosan-pEGFP nanoparticles were spherical,with an average size of(141.5 ± 26.7)nm,an average zeta-potential of(17.8 ± 3.9)mV and an average polydispersity index of(0.227 ± 0.025).The cytotoxicity test showed that the nanoparticles had a good biocompatibility with chondrocytes,quite different from LipofectamineTM 2000.The gene transfection in vitro proved that the nanoparticles were efficient in transfecting chondrocytes and expression of green fluorescent proteins was observed under fluorescent microscopy.The highest transfection efficiency(10.9％ ± 0.2％)was observed under the conditions of pH =7.0,N/P =5 and plasmid DNA dose =4.0 μg/mL.Conclusions Chitosan-pEGFP nanoparticles are a sort of effective non-viral gene transfer vector with potential transfection capacity against chondrocytes and little cytotoxicity.Closely dependent on pH value,N/P ratio and plasmid DNA dose,its transfection efficiency can reach the highest level when pH =7.0,N/P =5 and plasmid DNA dose =4.0 μg/mL.