The role of helper lipids in cationic liposome-mediated gene transfer
The role of helper lipids in cationic liposome-mediated gene transfer
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DOI：
10.1016/s0006-3495(96)79309-8
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发表时间：
1996-08-01
影响因子：
3.4
通讯作者：
Chan, K
中科院分区：
文献类型：
--
作者：
Hui, SW;Langner, M;Chan, K
在阳离子脂质体介导的转染过程中，阳离子脂质通常与“辅助脂质”混合以增加其转染效力。辅助脂质，包括二油酰磷脂酰胆碱（DOPC）和磷脂酰乙醇胺（二油酰PE），DO的重要性进行了检查。冷冻断裂电子显微镜的DNA：阳离子复合物含有pSV-β-GAL质粒DNA，阳离子脂质二油酰三甲基铵丙烷，和这些辅助脂质表明，最有效的混合物是ensheathed DNA和融合脂质体的聚集体。含PE的复合物迅速聚集时，添加到培养基中含有聚阴离子，而PC的复合物没有。然而，更多的颗粒的含PC的复合物形成后，复合物被添加到中国仓鼠卵巢（CHO）细胞在转染介质中的细胞表面。链霉蛋白酶处理抑制转染，而稀释的聚-L-赖氨酸增强转染，表明DNA的附着：脂质体复合物的细胞表面介导的静电相互作用。荧光光谱研究证实，更多的含PC的复合物比含PE的复合物与CHO细胞相关，并且随着时间的推移，更多的含PC的复合物位于低pH环境中（可能在内体内）。细胞松弛素B对PC脂质体转染的抑制作用强于PE脂质体转染。荧光标记的脂质和DNA摄取过程的共聚焦显微镜记录表明，许多颗粒的DNA：阳离子脂质体复合物作为一个整体被内化，而一些DNA聚集体被留在细胞表面后，脂质体的复合物与质膜融合。对于CHO细胞，内吞作用似乎是DNA：阳离子脂质体复合物的主要摄取途径。更多的含PC的颗粒比含PE的颗粒在细胞表面上形成的细胞色素细胞定向膜运动后，其各自的DNA：脂质体复合物附着到细胞表面通过静电手段。细胞表面颗粒的形成促进和/或触发内吞作用。阳离子脂质体和细胞膜之间的融合在确定转染效率中起次要作用。
In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a ''helper lipid'' to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.