The Effect Of Ultrasound On Apoptosis In Rat Vascular Smooth Muscle Cell Cultured In Vitro

Objective: To study the induction of apoptosis of rat vascular smooth muscle cell cultured in vitro irradiated with various intensity and exposure time ultrasound ,and to provide experimental bases for its clinical application in future.we observe the effects of ultrasound exposure on intracellular free calcium of rat vascular smooth muscle cell cultured in vitro,trying to seek if cell apoptosis by ultrasound induction mediates by intracellular calcium signaling. Methods: Rat vascular smooth muscle cells were cultured in vitro with DMEM. Cells were irradiated by ultrasound with dosage series and a frequency of 650 KHz . Apoptosis was detected 24 hours after irradiation by TUNEL marking method and electronic microscope respectively, Immunohistochemical method was used to evaluate the expressions of Bax;Concentrations of intracellular calcium in cultured smooth muscle cells were measured by calcium sensitive fluorescence indicator Fura-2-/AM technique. Results: 1 Apoptosis was detected 24 hours after irradiation by TUNEL marking method.The ratio of positive cells marked cell by TUNEL was 5.4%( 0.25W/cm2×2min) no more than in the controlled group 4.7% (P>0.05). The ratio of positive cells marked cell by TUNEL was 20.2%(1.0W/cm2×5min)and 15.6%(0.5W/cm2×5min) respectively higher than in the controlled group statistically(P<0.05). 2 Electron-microscopical observations:In experimental gorups(1.0W/cm2×5min and 0.5W/cm2×5min),many apoptotic phenomena were observed:electronic density of cytoplasm was high, showing many vesiculated and the form of apoptotic body,especially in 1.0W/cm2×5min group. 3 Bax protein expression:Except 0.25W/cm2×2min group,the positive rates of bax protein in experimental group were all increased than in the controlled group statistically (P<0.05). 4 In 0.25W/cm2×2min group,ultrasound increased [Ca2+]i in vascular smooth muscle cell in presence of calcium in medium ,but did not in absence of calcium in medium. In 0.5W/cm2×5min group,ultrasound increased [Ca2+]i in vascular smooth muscle cells in either presence or absence of calcium in medium,but [Ca2+]i was significantly higher (523.7±70.9nmol/L) presence of calcium than that (265.1±49.3nmol/L) in absence of calcium statistically (P<0.05). Conclusions: We found that ultrasound can induce apoptosis of rat vascular smooth muscle cells cultured in vitro in 1.0W/cm2×5min and 0.5W/cm2×5min group. In 1.0W/cm2×5min and 0.5W/cm2×5min group, the positive rates of bax protein in experimental group were all increased than in the controlled group (P<0.05).We observed that ultrasound stimulation increases the intracellular calcium concentration in vascular smooth muscle cells in 0.5W/cm2×5min group,including extracellular calcium influx and intracellular calcium release.These findings suggest that ultrasound induction of apoptosis of cell is associated with calcium signaling pathways that are activated in rat vascular smooth muscle cells cultured in vitro.