15. 超音波誘発アポトーシスに関する溶存気体および超音波造影剤レボビストの影響
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Although it has been known that ultrasonic cavitation induces reversible or irreversible cellular damage, the mechanism of apoptosis induced by ultrasound is still unclear. In this study, the effects of dissolved gases and an echo contrast agent, Levovist, on apoptosis induced by ultrasound were investigated. (Materials and Methods) Human histiocytic lymphoma U937 cells were maintained in RPMI 1640 culture medium containing 10% fetal calf serum. Sample solutions (3.5 ml) in polystyrene culture tubes were irradiated with continuous ultrasound at a frequency of 1 MHz and an intensity of 4.9 W/cm^2 (ISPTA). During sonication, the tubes were rotated at 30 rpm and the coupling water was maintained at 20℃. Apoptosis was examined by flow cytometry and fluorescence microscopy after staining with Annexin V-FITC/PI or Annexin V-Cy3. Free radical formation induced by ultrasound was studied by ESR-spin trapping with DMPO as the spin trap. (Results and Discussion) Apoptosis increased with the incubation time after sonication. Early apoptosis observed at 6 hrs after sonication reached its maximum at 1 min-sonication and gradually decreased. On the other hand, secondary necrosis increased with sonication time. The effects of dissolved gases, Ar, N2, O2, air, N2O, and CO2, were investigated. Free radical formation was found in solutions saturated with Ar, N2, O2, and air, but not with N2O and CO2. Apoptosis induced by ultrasound was dependent on the dissolved gases in the order Ar=N2=O2=air ≫ N2O=CO2〜0. These results suggest that ultrasound-induced apoptosis was due to acoustic cavitation accompanying free radical formation. An echo contrast agent, Levovist at a concentration of 50 mg/ml for 6 hrs induced apoptosis but no apoptosis was observed with less than 20 mg/ml. When the cells were sonicated in the presence of Levovist, enhancement of secondary necrosis induced by ultrasound was observed at concentrations of more than 20 mg/ml. In contrast, no increase of apoptosis was observed in the combined treatments. These results suggest that Levovist at high concentration, acting as cavitation nuclei, enhances secondary necrosis induced by ultrasound due to increase of the membrane damage. (Conclusion) Evidence of apoptosis in U937 cells induced by 1 MHz ultrasound was obtained. Apoptosis induced by treatment with Levovist for 6 hrs was observed only at high concentration. In addition, Levovist enhanced secondary necrosis but not apoptosis induced by ultrasound.
- 日本ソノケミストリー学会の論文
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- 15. 超音波誘発アポトーシスに関する溶存気体および超音波造影剤レボビストの影響