壳聚糖携载质粒纳米微球体外转染兔关节软骨细胞的实验研究

目的：研究携载质粒的不同分子量的壳聚糖纳米微球的包裹率和保护DNA的能力,镜下观察其大小和形态,观察其对原代兔关节软骨细胞的转染效率。方法：利用酶消化法消化3周龄新西兰大白兔的关节软骨,贴壁培养原代兔关节软骨细胞。购买相对分子量在5K和800K之间的八种壳聚糖,利用表达增强型绿色荧光蛋白的质粒（pEGFP）作报告基因,通过复合凝聚法制备壳聚糖-质粒纳米微球。琼脂糖凝胶电泳、紫外分光光度计分析不同N/P比值对不同分子量壳聚糖和质粒的结合能力及包封率的影响;纳米粒度仪、透射电子显微镜和环境扫描电子显微镜考察纳米微球的粒径分布和形态;荧光显微镜观察壳聚糖纳米微球介导pEGFP在体外培养的兔关节软骨细胞中的表达情况;流失细胞仪计算具体转染效率。结果：①N/P值为4及4以上时,各分子量的壳聚糖可完全包裹质粒成球;N/P值为2时,分子量为5K、50K、85K仅部分包裹质粒,其余可完全包裹;N/P值为1时,各壳聚糖均与质粒部分包裹;N/P值为0.25时,各壳聚糖均与质粒完全分离。②纳米粒度仪分析得出：N/P值为4时,各分子量的壳聚糖纳米微球的平均粒径均在1微米以下,③透射电子显微镜和扫描电子显微镜均可观察到球形或不规则形的大小不同的微球。荧光显微镜可大致观察到绿色荧光蛋白在软骨细胞内表达的表达情况。④流式细胞仪得出具体转染效率,分子量为170K、250K和800K的壳聚糖纳米微球的转染效率均高于5K、50K和85K的壳聚糖纳米微球,其中800K的壳聚糖纳米微球与脂质体相当（差异有统计学意义,P〈0.05）。结论：与脂质体相比,N/P比值为4时,相对分子量为800k的壳聚糖纳米微球可高效转染原代培养的兔软骨细胞,可以作为今后进一步体外、体内实验的首选转染载体。

Research on chitosan-pEGFP nanoparticles in transfection of primary rabbit articular chondrocytes

Objective：To investigate the DNA-loading effiency and the capability of protecting the plasmid of the nanoparticles with diffirent molecular weights,to observe their shape and size using electron microscope and to detect the transfection effiency of rab-bit articular chondrocytes in primary culture using chitosan-pEGFP nanoparticles.Methods：The articular cartilage of 3-week old rabbit was digested by diffirent enzymes,primary chondrocytes were cultured adherently in bottles.Eight kinds of chitosans with molecular weights between 5k and 800k were used.Chitosan-pEGFP nanoparticles were prepared by complex coacervation process with the report gene of plasmid expressing enhanced green florescence protein（pEGFP-C1）.The DNA-loading effiency of the nanoparticles with dif-firent molecular weights was detected by agarose gel electrophoresis and ultraviolet spectrophotometer when N/P ratio was changed.The particle size distribution was detected by nano zetasizer,and the shape was observed under transmission electron microscope and scan-ning electron microscopy.The expression of EGFP of CS-pDNA nanoparticle transfected chondrocytes was detected by florescence mi-croscope,and the transfection effiencies of different CS-pDNA nanoparticles were detected by flow cytometry.Results：①The plasmid can be inergrated totally with chitosans of all molecular weights when the N/P ratio was 4;The plasmid can be inergrated partially with chitosans of molecular weights of 5K,50K and 85K.and can be inergrated totally with others when the N/P ratio was 2;The plasmid can be inergrated partially with chitosans of all molecular weights when the N/P ratio was 1;The plasmid was totally detached with chitosans of all molecular weights when the N/P ratio was 0.25.② The average diameters of chitosan-pEGFP nanoparticles with all molecular weights were below 1μm.③ Diffirent shapes of sphere or irregular pattern were observed by transmission electron microscope and scanning electron microscopy.The high expression of EGFP in transfected chondrocytes could be observed under florescence micro-scope.④ Detected by flow cytometry,the transfection effiencies of CS-pDNA nanoparticles with molecular weights between 170k and 800k were higher than those with molecular weights between 5k and 85k significantly with P0.05,of which the CS-pDNA nanoparticle with molecular weight of 800k had the highest transfection effiency similar to positive control of Lipofectamine 2000.Conclusion：Com-pared with Lipofectamine 2000,transfection efficency of rabbit articular chondrocytes was high using CS-pDNA nanoparticle with molecular weight of 800k,which can be the first choice of the gene carrier in further transfection experiments in vivo or in vitro.