RESUMEN
PURPOSE: Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. METHODS: Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. RESULTS: A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. CONCLUSIONS: These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections.
Asunto(s)
Expresión Génica , Silenciador del Gen/efectos de los fármacos , Técnicas de Transferencia de Gen , Hígado/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transgenes , Animales , Tetracloruro de Carbono/farmacología , Ácido Clodrónico/farmacología , ADN/administración & dosificación , ADN/genética , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Hidrodinámica , Inyecciones Intravenosas , Interleucina-6/sangre , Interleucina-6/inmunología , Soluciones Isotónicas/administración & dosificación , Soluciones Isotónicas/farmacología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/patología , Liposomas , Hígado/inmunología , Hígado/patología , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Plásmidos/administración & dosificación , Plásmidos/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Rapid, large-volume injection, or so-called hydrodynamic injection, of naked plasmid DNA gives high transgene expression in mouse liver, and this method has been applied to liver-directed gene transfer in humans with slight modifications. To prove that injection-induced biological changes are involved in hydrodynamic injection-induced, high-level transgene expression in mouse liver, isotonic solutions were injected into mice that had received a hydrodynamic injection of plasmid DNA. Transgene expression in the liver was increased by such injections irrespective of the cDNA, promoter, and type of solution. This reactivation was repeatable and detectable even 3 months after gene transfer. Parameters required for reactivation were similar to those required for the hydrodynamic injection of plasmid DNA. Plasmid DNA-polyethyleneimine complex-based transgene expression in mouse liver was also reactivated by the same treatment. DNA microarray and quantitative RT-PCR analyses revealed that the expression of immediate-early response genes c-fos and c-jun was increased 70 and 100-fold, respectively. Activator protein (AP)-1- or nuclear factor (NF)-kappa B-dependent transgene expression was increased by an injection of isotonic solutions. These findings indicate for the first time that a rapid, large-volume injection of isotonic solution activates the transcription factors AP-1 and NF-kappa B in the liver, which in turn increases the transcription of genes delivered by hydrodynamic injection or other methods.