季铵盐壳聚糖及其纳米粒子作为基因载体的研究

摘要

The pDNA loading, protecting and transfection capacity of TMC and its nanoparticles were evaluated. The pDNA loaded nanoparticles were prepared by electrostatic interaction. The size, polydispersity, zeta potential and morphology of the nanoparticles were measured with dynamic laser scattering (DLS), atomic force microscope (AFM) and transmission electron microscopy (TEM). The protection of the nanoparticles for pDNA was observed by gel electrophoresis. The effect of the TMC/pDNA nanoparticles on cell viability was illustrated with MTT assay. Transfection activity of the nanoparticles was evaluated by gene transfection experiment in vitro in HepG 2 cells. Lipo-fectamine 2000 vector was used as control. The TMC/pDNA nanoparticles with a narrow size-distributed region and a regular spherical structure. TMC could partially protect the encapsulated plasmid pDNA from nuclease degradation as shown by electrophoretic mobility analysis. MTT assay shows that cell activity was not decreased remarkably. The gene transfection experiment in vitro suggested that TMC/pDNA nanoparticlesCnearly spherical shape,a particle diameter of 10 - 300 nm)could transfect HepG 2 cells effectively. When m(TMC)/m(pDNA) = 10 ∶ 1, the nanoparticles have the highest transfection efficiency at 48 h.%评价N,N,N-三甲基壳聚糖盐酸盐(TMC)及其纳米粒子对质粒DNA(pDNA)的负载及保护能力,考察了其纳米复合物对人类肝癌细胞株(HepG 2细胞)的转染能力.通过复凝聚法制备TMC/pDNA纳米粒子,并采用透射电镜及原子力显微镜表征粒子形态和粒径；采用凝胶阻滞分析观察其对pDNA的保护情况；采用四噻氮唑盐(MTT)比色法测定细胞毒性；以Lipofectamine 2000转染试剂作为阳性对照,检测其对HepG 2细胞的转染活性；采用荧光倒置显微镜观察转染情况.结果表明负载pDNA的TMC纳米粒子多呈球形,粒径为100～300 nm,能有效地包裹和保护基因不被DNaseⅠ酶消化；当TMC与pDNA的质量比为10∶1时,在48 h达到最高的转染效率.