聚酰胺-胺型树状聚合物介导基因转染的实验研究

Experimental Study of Gene Transfer Mediated by Polyamidoamine Dendrimers

目的探讨聚酰胺-胺型树状聚合物(PAMAM)作为基因载体,体外转染成纤维细胞的可行性。方法①将pEGFP与PAMAM按不同电荷比混合,通过原子力显微镜(AFM)对PAMAM和PAMAM-pEGFP复合物进行粒径、形态的表征;凝胶电泳实验和Zeta电位实验检验PAMAM与pEGFP质粒在不同电荷比时形成复合物的能力;通过DNA共沉淀试验检测不同电荷比下PAMAM对质粒pEGFP的包封率。②将不同电荷比的PAMAM-pEGFP复合物转染至小鼠胚胎成纤维细胞系,48 h后荧光显微镜下观察转染情况。③CCK-8实验检测转染后细胞的增殖活性。结果 PAMAM-pDNA复合物的粒径约10 nm。PAMAM在电荷比大于2时就能有效包裹质粒。随电荷比的增加,复合物的Zeta电位增加,在溶液中的稳定性和分散性也提高。包封率在电荷比为4:1时达95%。转染效率随电荷比的增加呈增高趋势,其中在4:1和6:1时转染效率达到最高。细胞毒性与电荷比有关,电荷比大于6:1时,PAMAM对细胞生长有毒性作用。结论 PAMAM纳米载体可安全有效地介导基因转染,是一种理想的基因递送系统。

Objective To explore the feasibility of PAMAM as a gene carrier transfecting into fibroblasts. Methods The p-EGFP was mixed with PAMAM by different charge ratios. ① The size and shape of PAMAM and PAMAM-pEG17P complex were characterized by Atomic force microscopy （AFM）. The ability of PAMAM forming complex was analysed by gel eleetrophoresis assay and Zeta potential measurement. The encapsulating efficiency was measured by co-sedimentation assay. ② The PAMAM-pEGFP complexes were delivered to mouse embryonic skin fibroblasts cell line （3T3）, and the transfection efficiency was observed after 48h by fluorescence microscope. ③ The post-transfeeted cell viability was determined by CCK- 8 test. Results The diameter of PAMAM-pDNA complex was 10 nm approximately. By a charge ratio of 2：1 or above, PAMAM can condense p-EGFP effectively. With the increase of charge ratio, the Zeta potential of the complex increased, the stability and dispersion of the complex in aqueous solution were also improved. By a charge ratio of 4：1, the envelopment rate can reach 95%. With the increase of charge ratio, the transfection efficiency increased, especially by a charge ratio of 4：1 or 6：1. The toxicity of the nanoparticles was dependent on charge ratio. When the charge ratio was over 4：l, the cell viability would be inhibited in certain extent. Conclusion PAMAM could transfect effectively and safely and becomes a promising gene delivery system.