猪肾细胞脂质磁转染系统的构建与表征

Construction and characterization of liposomal magnetofection system in pig kidney cells

摘要:

磁性纳米基因载体是一种非病毒基因载体，经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染，形成了一种新型动物体细胞转染方法，即称脂质磁转染(Liposomal magnetofection, LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统，本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合，用于携带外源基因转染动物体细胞。通过原子力显微镜 (AFM) 观测、ζ电位-粒度等分析表征手段，研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明，通过猪肾 (PK) 细胞的LMF实验，与脂质体 (Lipofectamine2000) 介导的转染比较，具有较高的转染率，更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。

Abstract:

Magnetic nano gene vector is one of the non-viral gene vectors, modified by functional group to bind cationic transfect reagents. Coupling magnetofection with the universal lipofection we developed a novel somatic cell transfection method as the so-called liposomal magnetofection (LMF). This approach is potential to provide somatic cell cloning with stable genetic cell lines to cultivate transgenic animals. In order to construct such liposomal magnetic gene vectors complexes system, we used nano magnetic gene vector to combine with liposomal cationic transfect reagents by molecular self-assembly. This vectors system successfully carried exogenous gene and then transfected animal somatic cells. Here, we conducted atomic force microscopy (AFM), zeta potential-diameter analysis and other characterization experiments to investegate the size distribution and morphology of magnetic nanoparticles, the way of the vectors to load and concentrate DNA molecules. Our data reveal that, the LMF of Pig Kidney cells exhibited higher transfection efficiency comparing with the transfection mediated by the commercial lipofectamine2000. Moreover, LMF method overcomes the constraint of transient expression mediated by lipofection. Meanwhile, MTT assay showed low cytotoxicity of LMF. Hence, LMF is a feasible, low cytotoxic and effective method of cell transfection.