聚乙烯亚胺包裹小环DNA形成的纳米颗粒传输外源基因的性质表征

Polyethylenimine and minicircle DNA based gene transfer

摘要:

聚乙烯亚胺 (PEI) 是一种具有良好生物安全性和生物相容性的非病毒载体，能高效转染肿瘤细胞。小环DNA是一种去除质粒细菌骨架，只含有目的基因表达框的环状DNA分子。与普通质粒相比，小环DNA具有表达效率高、持续时间长的优势。使用PEI包裹携带报告基因gfp和抑癌基因pten小环DNA载体，并利用各种技术手段分析了该传输系统的理化性质和生物学效应。凝胶阻滞实验、电镜实验及MTT实验分析结果表明利用PEI包裹小环DNA和质粒DNA体系性质无显著的差别，并且2种复合物对细胞毒性亦无明显差别；但是动态光散射实验结果显示由于PEI可以包裹更多数量的小环DNA，所以PEI包裹小环DNA形成的复合物粒径要略大于包裹质粒DNA形成的复合物粒径。荧光显微镜实验、real-time PCR分析和Western blotting 分析结果表明，PEI包裹小环DNA形成的复合物对细胞的转染效率要远远高于PEI包裹质粒DNA所形成的复合物，并且小环所携带的外源基因的表达效率要远远高于质粒DNA所携带的外源基因的表达效率。实验结果表明，PEI包裹小环DNA形成的纳米颗粒在细胞转染过程中具有很高的表达效率，这一研究结果为PEI包裹小环DNA的非病毒载体系统在传输外源基因过程中的应用提供理论基础和技术支持。

Abstract:

Polyethylenimine (PEI) is one of the most characterized non-viral vectors. It can condense DNA in a good manner and achieve high transfection efficiency. Minicircle DNA (mc-DNA) is a novel kind of supercoiled DNA which is devoid of bacterial backbone. mc-DNA is superior to conventional DNA for its higher transfection effciciency and longer time-span. In this study, we combined PEI and mc-DNA in gene delivery system. We investigated the physicochemical and biochemical effects of this non-viral system and further explore its potential in tumor gene therapy. mc-DNA was obtained by recombination of parental plasmid in the presence of L-arabinose, and complexed with PEI. The results of transmission electron microscopy and scanning electron microscopy showed that the particles were spherical and homogeneous. Through gel retardation assay and MTT assay, we found that there were no obvious differences in binding capability of PEI to mc-DNA and plasmid DNA, as well as in cytotoxicity. The results of dynamic light scattering showed that the size of PEI/mc-DNA was about 68 nm, a slight larger than that of PEI/plasmid DNA. Furthermore, the tumor cells transfected with mc-GFP showed higher GFP expression level than that of conventional plasmid. The same results were achieved in the cells treated with tumor-suppressor gene pten, assayed by RT-PCR and Western blot. It indicates that the system of PEI/minicircle DNA is promising in gene transfer.