Rémy-Kristensen A, Clamme J P, Vuilleumier C, Kuhry J G, Mély Y
Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, BP 24, 67401 Illkirch Cedex, France.
Biochim Biophys Acta. 2001 Sep 3;1514(1):21-32. doi: 10.1016/s0005-2736(01)00359-5.
Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.
聚乙烯亚胺（PEI）是基因治疗中最有效的非病毒载体之一。本研究的目的是探讨内吞作用在PEI/DNA复合物转染同步化L929成纤维细胞中的作用。这是通过共聚焦显微镜和流式细胞术进行的，使用内吞作用标记物FM4-64以及用DNA嵌入剂YOYO-1或与PEI共价连接的荧光素标记的PEI/DNA复合物。内吞作用似乎是PEI/DNA复合物进入L929细胞的主要（如果不是唯一）方式。这些复合物遵循典型的液相内吞途径，并在不到10分钟的时间内有效地被直径不超过200 nm的内体摄取。随后，复合物的定位变为核周，并且显示晚期内体之间发生融合。与EA.hy 926细胞中相同复合物的细胞内运输进行比较（W.T. Godbey、K. Wu、A.G. Mikos，《美国国家科学院院刊》96 (1999)）表明，PEI/DNA复合物的内吞作用强烈依赖于细胞。在L929细胞中，复合物从内体中逃逸是转染的主要障碍。这将转染细胞的数量限制在百分之几，尽管在大多数细胞中都观察到了PEI/DNA复合物的内化。此外，复合物进入细胞核显然需要有丝分裂，并且不涉及内体膜的脂质。这种进入似乎是一个短暂的事件，只涉及少数复合物。
