目的:以增强型绿色荧光蛋白质粒(pEGFP)为报告基因,探讨影响壳聚糖(CS)介导的基因转染率的因素。方法:复凝聚法制备pEGFP/CS纳米粒,选用HEK293细胞,考察CS相对分子质量、CS中的氨基与pEGFP中的磷酸基的比值(N/P比)、纳米粒粒径、介质pH值和血清对转染率的影响。结果:相对分子质量5×10^3的CS获得较高转染率;转染率随N/P比增加而提高;介质pH≤6.8有利于转染;在230~1290nm范围,粒径对转染效率无影响;CS介导的转染不受血清影响。结论:CS相对分子质量、N/P比和介质pH值是影响转染率的因素,低相对分子质量水溶性CS有利于提高转染。
Objective:To investigate the factors affecting in vitro transfection efficiency mediated by chitosan (CS) using the enhanced green fluorescent protein plasmid (pEGFP) as reporter gene. Methods:pEGFP/CS nanopanicles were prepared by the complex coacervation method. The effects of the molecular weight of CS,N/P ratio, panicle size of the uanoparticles, pH of transfection medium and the presence of serum on transfection efficiency were investigated in HEK293 cells. Results:The transfection efficiency of pEGFP/CS nanoparticles was found to be highest when the molecular weight of chitosan was 5 KDa or the pH of transfection medium was no more than 6.8. Moreover,the transfection efficiency was independent on the particle size in the range of 230 to 1 290 nm and was enhanced by the increase of N/P ratio. Furthermore, the presence of serum had no impact on the CS-mediated transfection efficiency. Conclusion: The molecular weight of CS, N/P ratio and pH of transfection medium were the important factors which would affect transfection efficiencies mediated by CS. Low molecular weight and watersoluble CS was advantageous to improving transfection efficiency.