以聚乙烯亚胺为骨架的非病毒基因载体的构建及其转染大鼠肝细胞的研究

目的 构建交联聚乙烯亚胺（Polyethylenemine,PEI）衍生物PEI-Bu,研究其对大鼠肝细胞系BRL-3A的细胞毒性和转染效率.方法 以PEI 800Da为骨架,1,4-丁二醇二氯甲酸酯为连接剂制备聚合物PEI-Bu,用凝胶渗透色谱法（GPC）测定分子量,动态光散射法（DLS）测定PEI-Bu/pDNA复合物的粒径和Zeta电位,琼脂糖凝胶电泳考察其复合质粒DNA的能力.MTT法榆测PEI-Bu对BRL-3A的细胞毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/pDNA复合物在BRL-3A细胞中的转染效率.结果 GPC 测定分子量为Mw4289Da,多分散指数1.31,复合物的粒径为138-16lnm,Zeta电位为2.4-4.3mV,凝胶电泳表明在质量比大于1时 PEI-Bu 具有复合DNA的能力,在同一浓度下PEI-Bu的细胞的毒性小于PEI 25kDa （p〈0.01）,PEI-Bu/pDNA在质量比为5时达到最高转染效率,高于PEl25kDa （p〈0.01）.并与Lipofectamine2000 相当 （P〉0.05）.结论 PEI-Bu对BRL-3A 细胞是一种低细胞毒性、高转染效率的非病毒基因载体,在肝细胞基因治疗领域中具有潜在的应用前景.

Construction of non-viral gene carrier based on polyethylenemine and its transfeotion in rat liver cells

Objective To synthesize cross-linked Polyethylenimine derivative PEI-Bu and investigate its cytotoxicity and transfection efficiency in rat liver cell line BRL-3A. Methods PEI-Bu was synthesized by using PE1800 Da as a backbone and 1, 4-Dibutanediol Chloroformate was used as a linker. The molecular weight of the polymer was measured by gel per- meation chromatography （GPC）. The particle size and zeta potential of complexes were measured by dynamic light scattering （DLS）. The pDNA condensation ability of the polymer was evaluated by agarose gel electrophoresis. MTT assay was em- ployed to measure the cytotoxicity of the polymer. Luciferase plasmid was used as the reporter gene to investigate the trans- fection efficiency in BRL-3A cells. Results The molecular weight （Mw） of the polymer measured by GPC was found to be 4289Da, with a polydispersity index of 1.31. The particle size of polymer/pDNA complexes was between 138nm and 161nm, zeta potential was 2.4-4.3mV. Gel retardation assay showed complete condensation ofpDNA at w/w ratios exceeded 1. Cytotoxicity of PEI-Bu was lower than PEI 25kDa at the same concentration. Transfection result indicated that the polymer performed the highest transfection efficiency at w/w 5, which was higher than commercially available PEI 25 kDa （P〈0.01） and comparable with Lipofectamine 2000 （P〉0.05）. Conclusions The polymer PEI-Bu displays much lower cytotoxicity and significantly enhances transfection efficiency than PEI 25kDa in BRL-3A cells. Therefore, it would be a promising candidate in hepatocyte gene therapy.