线性聚乙烯亚胺(LPEI)介导DNA转染的优化研究

Optimization of DNA Transfection Mediated by Linear Polyethylenimine (LPEI)

线性聚乙烯亚胺(LPEI)是一类广泛用于核酸转染或药物传递的阳离子聚合物.为了进一步探究25kDaLPEI的转染条件,通过凝胶电泳阻滞试验评估N/P,聚合pH及聚合时间对LPEI-DNA复合物形成的影响,继而利用大范围N/P的25kDaLPEI对HEK293T细胞进行绿色荧光蛋白表达质粒转染,转染48h后通过绿色荧光细胞比例及MTT的统计分析,评估转染效率与LPEI造成的细胞毒性.结果表明,N/P的上升能增强LPEI-DNA的电泳阻滞;随着共孵育pH值从6~8升高,LPEI-DNA聚合逐渐减弱;随着共孵育时间延长,LPEI-DNA聚合逐渐增强,孵育1h时达到饱和.随着体外转染HEK293T细胞的N/P升高,转染阳性细胞比例逐渐升高,当N/P达到40~60时转染效率达到最高,进一步提高N/P后转染效率极显著下降(p<0.01);随着N/P升高,细胞活力逐渐下降.结果表明,N/P,pH,聚合时间均可影响LPEI聚合核酸能力;综合考虑N/P对HEK293T细胞转染效率及细胞毒性的影响,确定25kDaLPEI转染细胞适宜的条件为N/P为40~60,聚合pH值为6.0,聚合时间为1h.

Linear polyethylenimine (LPEI) is a cationic polymer which is widely used for nucleic acid transfection and drug transport. In order to further explore the transfection conditions of25 kDa LPEI,gel retardation assay was used to investigate the combined effect of N/P,co-incubation time of LPEI and DNA and co-incubation pH of LPEI-DNA on the formation of LPEI-DNA complex in this research. Then HEK293 T cell was used to transfect the 25 kDa LPEI with a GFP expression plasmid in a wide range of N/P,and the transfection efficiency and cytotoxicity were assessed with statistic analysis of fluorescent cell percent and MTT after 48-h transfection. The results showed that the gel retardation of LPEI-DNA increased with N/P,LPEI-DNA polymerization gradually decreased with the increase of co-incubation pH from 6 to 8,and LPEI-DNA polymerization gradually increased with the extension of co-incubation time and reached saturation at1 h. With the increase of N/P in in vitro transfected HEK293 T cells,the proportion of transfected positive cells gradually increased. When N/P reached 40-60,the transfection efficiency reached the maxximum. With further increase in N/P,the transfection efficiency sharply decreased ( p <0.01). The cell vabiliity gradually decreased with increasing N/P. Conclusion: N/P,polymerization pH and polymerization time all affected the ability of25 kDa LPEI to polymerize nucleic acid. Considering the effect of N/P on HEK293 T cell transfection efficiency and cytotoxicity,the optimal conditions for25 kDa LPEI transfection were N/P=40-60,polymerization pH=6.0,and polymerization time =1 h.