聚乙二醇－聚乙烯亚胺介导小干扰RNA体外转染神经干细胞的研究

Delivery of small interfering RNA into neural stem cells mediated by polyethylene glycol- polyethyleneimine in vitro

目的研究聚乙二醇－聚乙烯亚胺（PEG．PEI）作为非病毒纳米载体介导小干扰RNA（siRNA）体外转染C17．2 NSCs的效果。方法采用自行设计合成的PEG．PEI与靶向Nogo受体复合体基因的siRNA形成复合物，通过粒径与电位的测定、凝胶阻滞电泳实验等方法观察PEG—PEI／siRNA纳米复合物的表征及复合效果。以脂质体复合体系Lipofectamine 2000／siRNA为对照，采用流式细胞仪检测不同N／P比（PEG—PEI中氮原子和siRNA中磷原子的摩尔比）的PEG－PEI／siRNA复合物对NSCs的转染效率。结果PEG－PEI和siRNA形成粒径为纳米级别的复合物，随着N／P比增大，复合物的粒径逐渐减小，而表面电位逐渐增大。凝胶阻滞电泳实验表明siRNA与PEG—PEI可以通过静电相互作用而稳定结合。流式细胞仪检测细胞转染率后发现，N／P=15时，PEG－PEI／siRNA复合物转染率最高，可达（78．72±8．18）％。结论PEG－PEI是一种有良好发展前景的非病毒型siRNA转运载体，对神经干细胞基因治疗具有潜在的应用价值。

Objective To explore the effect of polyethylene glycol-polyethyleneimine （PEG-PEI） serving as a non-viral vector in delivering small interfering RNA （siRNA） into C17.2 neural stem cells （NSCs） in vitro. Methods Complexes of PEG-PEI and siRNA targeting Nogo receptor were prepared, and their characterizations were estimated by measurements of particle size and zeta potential, and the complex abilities of PEG-PEI/siRNA complexes were observed by gel retardation assay, In addition, with liposome complex system （Lipofectamine 2000/siRNA） as positive control, the transfection efficiency of PEI-PEG/siRNA complexes at different N/P ratios （cationic nitrogern/siRNA phosphate molar ratio） was detected by flow cytometry. Results The siRNA molecules were condensed by PEG-PEI to form nanoscale complexes. As the proportion of N/P ratio enhancing, the surface potential of nanoparticles gradually increased and the particle sizes of PEI-PEG/siRNA complexes showed a decreasing trend. Gel retardation electrophoresis suggested that siRNA could be fully composited with PEG-PEI as a result of the coulombic force between them. Meanwhile, flow cytometry experiments revealed that the transfection efficiency of PEG-PEI mainly depended on N/P ratios of the nanoparticles, and the highest one was obtained at N/P=I5 （[78.72±8.18）] %）. Conclusion PEG-PEI might be a prospective candidate for siRNA delivery system, which enjoys its value in NSC gene therapy.