新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.

Study on a Novel Non-virus Triplex Gene Vectors Composed of Plasmid DNA

In order to study the feasibility and transfection mechanisms of a novel gene vector system composed of plasmid DNA-anti DNA antibody-cationic lipid(DAC)for non-virus site-specific gene therapy,stable DAC triplex nano-micelles were formed in PBS system through self-assembling of plasmid DNA,anti DNA antibody and cationic lipid.The mechanisms of cell uptake and entry with DAC were observed by fluorescent confocal microscopy.Transfection efficiency of DAC was estimated by using A10 cell in vitro with GFP-DNA(green fluorescent)triplex micelles.Cell culture studies revealed that DAC triplex micelles markedly increased the level of gene transferred to A10 cells,with more than 4-fold increase in transfection compared to DC group without anti DNA antibody and 11-fold increase compared to DA group without cationic lipid.Fluorescent DNA studies demonstrated greater cell uptake in vitro with DAC compared to the control formulations.Confocal microscopy studies confirmed nuclear entry in A10 cells with DAC,while formulations without anti-DNA antibody demonstrated no nuclear entry.It can be concluded that plasmid DNA-anti DNA antibody-cationic lipid(DAC)triplex micelle was a novel non-viral gene vector with high transfection efficiency and no cytotoxicity.It was hypothesized that DAC triplex micelles could enhance DNA delivery through mechanisms involving:increased levels of DNA incorporation into micelles due to antibody binding,enhanced plasmid DNA entry into nuclear due to the nuclear entry properties of anti-DNA antibody,and increased transfection efficiency via co-operation of anti-DNA antibody and cationic lipid.Gene delivery using DAC triplex micelles should be suitable for a wide array of single or multiple therapeutic gene strategies and for further successful gene therapy.