P17 細胞膜修飾による超音波遺伝子導入の増強(物質創製、他)

抄録

Mechanism of ultrasound-mediated transfection has not yet been completely elucidated, though, it is considered that mechanical stress generated with inertial cavitation may be playing a pivotal role and that cells may uptake DNA while cell membrane is repaired after disruption by the stress, resulting in transfection. Since cell membrane is the first and possibly the most important barrier for establishing the transfection, we examined effects of known membrane modifiers such as a local anesthetic, lidocaine, and different temperatures on the transfection. We observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44 ℃) on USMGT (1 MHz, 3.6 W/cm^2 (I_<SATA>) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope. Lidocaine (1 mM) and heat (42-44℃) significantly increased luciferase expression approximately 18-fold and 19-fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells. These results indicated that not only strengthening cavitation occurrence but facilitating modification of cell membrane are useful for enhancing transfction efficiency that has been pointed out to be modest in the case of the one by ultrasound.