Sommer W, Cui X, Erdmann B, Wiklund L, Bricca G, Heilig M, Fuxe K
Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
Antisense Nucleic Acid Drug Dev. 1998 Apr;8(2):75-85. doi: 10.1089/oli.1.1998.8.75.
The fate of 15-mer phosphorothioate-modified antisense oligonucleotides to c-fos was followed after their microinjection into rat brain. Using radiolabeled oligonucleotides, it was demonstrated that the bulk of the material stays in the injected region but that a minor part is transported with the projection pathways to regions far away from the site of injection. Using tetramethylrhodamine-isothiocyanate (TRITC) labeling as well as fluorescein isothiocyanate (FITC) labeling, it was found that the oligonucleotides were taken up by a great number of cells within 30 minutes after the injection. A diffuse cytoplasmic staining and also nuclear staining were observed in these cells, which could be identified exclusively as neurons by double labeling for the neuron-specific protein NeuN. At later times (6, 24, and 48 hours), the appearance of the oligonucleotides changed gradually to a punctate cytoplasmic staining, which by electron microscopic analysis was shown to be caused by the presence of the oligonucleotides in intracellular vesicles. The pattern of intracellular fluorescence was changed when the oligonucleotides were injected together with the cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). A small number of astrocytes and microglial cells were found to be labeled by the oligonucleotides, but only at later times after the injection and exclusively in a punctate cytoplasmic manner. Thus, the uptake of oligonucleotides in the nerve and glial cell populations of the brain might involve different mechanisms, the one in the neurons appearing to be very rapid and potent.
将15聚体硫代磷酸酯修饰的针对c-fos的反义寡核苷酸显微注射到大鼠脑内后，对其命运进行了追踪。使用放射性标记的寡核苷酸，结果表明大部分物质留在注射区域，但有一小部分通过投射通路转运到远离注射部位的区域。使用异硫氰酸四甲基罗丹明（TRITC）标记以及异硫氰酸荧光素（FITC）标记，发现在注射后30分钟内，大量细胞摄取了寡核苷酸。在这些细胞中观察到弥漫性的胞质染色以及核染色，通过对神经元特异性蛋白NeuN进行双重标记，这些细胞可明确鉴定为神经元。在随后的时间点（6、24和48小时），寡核苷酸的外观逐渐变为点状胞质染色，通过电子显微镜分析表明这是由于细胞内囊泡中存在寡核苷酸所致。当寡核苷酸与阳离子脂质1,2-双（油酰氧基）-3-（三甲基铵）丙烷（DOTAP）一起注射时，细胞内荧光模式发生了变化。发现少量星形胶质细胞和小胶质细胞被寡核苷酸标记，但仅在注射后的较晚时间，且仅以点状胞质方式标记。因此，大脑神经和胶质细胞群体对寡核苷酸的摄取可能涉及不同机制，其中神经元中的摄取似乎非常迅速且有效。
