交联聚乙烯亚胺衍生物的构建及其转染COS-7细胞的研究

Construction of Cross-linked Polyethylenimine Derivative and Its Transfection in COS-7 Cells

目的:优化构建交联聚乙烯亚胺(Polyethylenemine,PEI)衍生物PEI-Bu,研究其对非洲绿猴肾成纤维细胞系(COS-7)的转染活性和细胞毒性。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,琼脂糖凝胶电泳考察其复合质粒DNA的能力,MTT法检测PEI-Bu对COS-7的毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/DNA复合物在COS-7细胞的转染活性。结果:凝胶电泳表明PEI-Bu/DNA在质量比大于1时即具有复合DNA的能力,PEI-Bu的细胞毒性随浓度增大而增大,在同一浓度下PEI-Bu的细胞毒性小于PEI 25kDa,(P<0.05),PEI-Bu/DNA在质量比为5时达到最高转染活性,高于PEI 25kDa(P<0.01),并与Lipofectamine2000相当(P>0.05)。结论:PEI-Bu在COS-7细胞中是一种低细胞毒性、高转染活性的非病毒基因载体(与商业化的PEI 25kDa比较),其在基因治疗领域中具有潜在的应用前景。

Objective： To synthesize cross-linked Polyethylenimine derivative with modified procedure and investigate its cytotoxicity and transfection efficiency on COS-7 cells.Methods： PEI-Bu was synthesized by using PEI 800Da as a backbone and 1,4-Dibutanediol Chloroformate was used as a linker.The pDNA condensation ability of the polymer was evaluated by agarose gel electrophoresis.MTT assay was employed to measure the cytotoxicity of the polymer.Luciferase plasmid was used as the reporter gene to investigate transfection efficiency in COS-7 cells.Results： Gel retardation assay showed complete condensation of pDNA at w/w ratios exceeded 1.Cytotoxicity of PEI-Bu increased with the increasing of the concentrations.PEI-Bu showed lower cytotoxicity 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 that in commercially available PEI 25kDa（P〈0.01） and comparable with Lipofectamine 2000（P〉0.05）.Conclusion： The polymer PEI-Bu displayed much lower cytotoxicity and significantly enhanced transfection efficiency than PEI 25kDa in COS-7 cells,Therefore,it would be a promising candidate for safe and efficient gene delivery in gene therapy.