Keller H, Schu B, Rüdel R, Brinkmeier H
Department of General Physiology, University of Ulm, Ulm, Germany.
J Pharmacol Exp Ther. 2000 Oct;295(1):367-72.
The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na(+) channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 microM, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na(+) currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells.
针对两种密切相关的钠通道亚型hSkM1或hH1的RNA专门设计的15聚体硫代磷酸反义寡脱氧核苷酸（aODNs）的作用，在人肌管中进行了测试。荧光（3'-异硫氰酸荧光素）标记显示，仅将培养物与aODNs孵育不会导致aODNs的摄取，但脂质体介导的转移是成功的，并导致ODNs在细胞质和细胞核中定位。细胞内荧光至少稳定3天。在5 microM时，hH1特异性aODN可有效抑制离子通道功能，但hSkM1特异性aODN则无效。逆转录-聚合酶链反应在mRNA水平上给出了相应的结果。然而，在稳定表达hSkM1的HEK-293细胞中，相同的hSkM1特异性aODN能够降低钠电流（2.4±0.5 nA，n = 11；对照组：6.5±1.0 nA，n = 14）。我们得出结论，aODNs的细胞摄取和RNA靶标的细胞内进入是必要的，但并非总是完整细胞中有效阻断mRNA翻译的充分条件。
