Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
Department of Cytopathology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
Bioelectrochemistry. 2015 Jun;103:111-9. doi: 10.1016/j.bioelechem.2014.08.020. Epub 2014 Aug 29.
Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.
电脉冲（电穿孔/电渗透）的应用是体外和体内基因转移（即基因电转移（GET））的有效方法。目前，DNA 进入细胞的机制尚未完全了解。实验证据表明，内吞作用是 DNA 进入细胞后表达的主要机制。因此，我们的研究目的是阐明内吞作用抑制剂甲基-β-环糊精（MβCD）、刀豆球蛋白 A（ConA）和 Dynasore 是否可以损害体外 B16F1 鼠黑色素瘤和体内小鼠 m. tibialis cranialis 中 GET 的转染效率。我们表明，MβCD——一种通用的内吞作用抑制剂——几乎可以阻止 EGFP-N1 质粒的体外 GET，ConA——网格蛋白介导的内吞作用抑制剂——也会阻断 GET，但程度较小，而当使用 Dynasore——一种可逆的动力蛋白抑制剂——仅在 GET 后阻断内吞作用 5 分钟时，对 GET 效果没有影响。此外，MβCD 还降低了体内 m. tibialis cranialis 中的 GET 效率，这种效果是持久的。本研究结果表明，内吞作用可能是体外和体内 GET 后 DNA 进入的主要机制。
