论文部分内容阅读
研制无毒、高转染效率的聚乙烯亚胺(PEI)阳离子纳米粒,Zeta粒度仪、紫外可见光谱表征纳米粒,琼脂凝胶电泳阻滞实验分析报告基因质粒pEGFP-N1与PEI及聚乙二醇(PEG)修饰后PEI的复合能力及抗DNAseI酶降解能力,MTT检测纳米粒的细胞毒性,体外转染试验及流式细胞仪评价纳米粒的体外转染活性。以pEGFP-N1质粒与PEI阳离子纳米粒的电荷比(N/P)、PEG与PEI的接枝比例和质粒转染剂量为条件,转染效率为指标,正交设计法优化pEGFP-N1/PEI-PEG阳离子纳米粒介导的肝癌细胞基因转染效率。结果显示,PEG修饰后的PEI纳米粒对肝癌细胞毒性较小,粒径及电位较稳定,对质粒有较好的复合能力和抵制DNAseI酶降解能力;pEGFP-N1/PEI的N/P比为40∶1、基因质粒剂量为每孔6μg时,纳米粒复合物的转染效率最高为91%。PEG修饰后的PEI是一种高效、无毒的非病毒载体,能有效将基因转染入肝癌细胞,优化实验条件可提高转染效率。
Polyethyleneimine (PEI) cationic nanoparticles with non-toxic and high transfection efficiency were developed. Zeta particle size analyzer and UV-Vis spectrum were used to characterize the nanoparticles. The agarose gel electrophoresis was used to analyze the expression of reporter gene plasmid pEGFP-N1, PEI and poly The ability of PEI modified by diol (PEG), the capability of degrading DNAseI, the cytotoxicity of nanoparticles by MTT, the in vitro transfection assay and the in vitro transfection activity of nanoparticles by flow cytometry. The efficiency of pEGFP-N1 / PEI was optimized by orthogonal design with the charge ratio (N / P) of pEGFP-N1 plasmid and PEI cationic nanoparticle, the graft ratio of PEG to PEI and the transfection dose of plasmid. -PEG cationic nanoparticles mediated gene transfection efficiency of hepatocellular carcinoma cells. The results showed that the PEG-modified PEI nanoparticles were less toxic to liver cancer cells, the particle size and potential were more stable, and had better complex ability against plasmids and resistance to DNAseI enzyme degradation. The N / P ratio of pEGFP-N1 / PEI 40: 1, the highest plasmid transfection efficiency of nanoparticle composites was 91% at the dose of 6μg / well. PEG-modified PEI is an efficient, non-toxic non-viral vector, which can effectively transfect the gene into liver cancer cells. Optimizing the experimental conditions can improve the transfection efficiency.