Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes
Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes
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DOI：
10.1016/0005-2736(95)00219-7
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发表时间：
1996-01-12
影响因子：
3.4
通讯作者：
Debs, RJ
中科院分区：
文献类型：
--
作者：
Friend, DS;Papahadjopoulos, D;Debs, RJ
阳离子脂质体介导哺乳动物细胞的高效转染，但细胞内化和处理阳离子脂质体- dna复合物的方式尚未得到很好的表征。我们将几种细胞类型，包括人类和小鼠红细胞白血病细胞，非洲绿猴肾细胞（cv1），分离的大鼠肺泡II型细胞和肺泡巨噬细胞暴露于含有N-（1-2,3-二聚氧丙基）-N，N，N-三乙基铵（DOTMA）和二油基磷脂酰乙醇胺（DOPE）的dna阳离子脂质体复合物中。通过电子显微镜观察脂质体与细胞相互作用的形态。脂质体制剂可与胶体金颗粒或质粒DNA和金颗粒复配。经DOTMA脂质体- dna复合物处理的细胞在包被凹坑中表现出脂质体- dna复合物的内吞作用，可见于早期核内体、晚期核内体和溶酶体。在离体肺泡II型细胞中，金标记的DOTMA脂质明显与板层体内容物混合。在大多数细胞中，金颗粒分散在细胞质基质中。在一小部分cv - 1和U937细胞中，在细胞核内发育了类似内质网的膜系统。从CV-1细胞中分离出细胞核并与含有dotma的脂质体混合后，这种新结构也出现在细胞核中。暴露于含有dotma的脂质体后形成的膜厚度为10纳米，与约10纳米相比。内源性细胞膜厚度为8 nm。基于这些形态学观察，我们提出阳离子脂质体进入细胞的主要途径是内吞作用。在某些情况下，内体室释放其阳离子脂质体dna内容物到细胞质基质中。偶尔，脂质体可以通过与核膜融合进入细胞核，形成囊泡和网状核膜。目前尚不清楚，如果任何这些形态学观察与阳离子脂质体介导的转染有关。
Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells, African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-I and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA-containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes.