非病毒载体介导的siRNA细胞内导入机理研究

比较了两类非病毒载体,包括阳离子聚合物载体(HPAMAM-PHE45,PEI)和阳离子脂质载体(DOTAP)运载siRNA进入细胞的动态过程.DOTAP/siRNA能够很快地被内吞进入内涵体途径,6 h内就能将绝大多数的siRNA从内涵体中释放出来.而HPAMAMPHE45,PEI/siRNA复合物在与细胞接触0.5 h后首先附着在细胞膜表面,逐渐地部分复合物也经由内涵体途径将siRNA从内涵体中释放出来进入细胞质.但也有一部分复合物可能从窖蛋白介导的内吞途径进入细胞.实验结果显示,细胞摄取阳离子脂质/siRNA主要通过网格蛋白介导的内吞实现,而阳离子聚合物/siRNA主要通过网格蛋白介导的内吞和窖蛋白介导的内吞共同完成.这两种不同的内吞方式可能对非病毒载体的不同转染机理及优化理论有较大影响.深入研究其中的关键因素可以为非病毒载体运载siRNA内吞和胞内途径的机理提供线索.

Examination of the dynamic cellular internalization processes of siRNA mediated by nonviral vectors

The study investigated the dynamic process of internalization and intracellular processing of DOTAP/siRNA Lipoplexes and PEI/ siRNA, HPAMAM-PHE45/siRNA Polyplexes by H1299 cells. Fluorescence colocalization studies with a lysosomal marker-LysoTracker, revealed that DOTAP complexes could be quickly take up by cells into the cytoplasm, and most were released from endosomal pathway by 6 h. HPAMAM-PHE45 or PEI complexes were found to attach to the surface of the cell membrane initially. Some of the complexes followed the endosomal pathway, but also a part of complexes enter cells through caveolae-mediated endocytosis. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while Polyplexes are taken up by two mechanisms, involving caveolae and clathrin-mediated endocytosis. In this study, we compared the different siRNA uptake and intracellular processing of Polyplexes and Lipoplexes delivery systems basis optimization study in the future.