超声联合微泡诱导血管平滑肌细胞凋亡的机制研究

Mechanism of apoptosis of vascular smooth muscle cells induced by ultrasound irradiation combined with microbubbles

目的探讨低频超声联合脂质微泡诱导血管平滑肌细胞(VSMCs)凋亡的可能机制。方法以低频连续波超声(频率1MHz、声强0.3W/cm2)联合脂质微泡(1μl/ml)辐照VSMCs120s。分别用流式细胞仪Annexin V/PI染色、免疫细胞化学技术、荧光探针Fura-4/Am负载后激光共聚焦法检测细胞凋亡、凋亡相关蛋白表达和细胞内游离Ca2+浓度的变化情况。结果与对照组和血小板衍生生长因子-BB刺激组(PDGF)比较,两组超声辐照联合微泡[(US+MB)组和(PDGF+US+MB)组]的VSMCs凋亡率显著增加、Bax蛋白表达明显上调、而Bcl-2蛋白表达则显著减少(P<0.01);而且PDGF+US+MB组的细胞凋亡率明显高于US+MB组,Bax蛋白表达上调和Bcl-2蛋白表达下调的程度较US+MB组更为显著(P<0.01)。各实验干预组VSMCs内Ca2+浓度均较对照组显著增高(P<0.01);PDGF+US+MB组和US+MB组细胞内Ca2+浓度均明显低于PDGF组(P<0.01)。结论超声联合微泡诱导VSMCs凋亡的机制可能与细胞内游离Ca2+浓度增加、Bax蛋白表达增加和Bcl-2蛋白表达降低有关。

Objective To investigate the mechanism of vascular smooth muscle cells （VSMCs） apoptosis induced by ultrasound irradiation combined with liposome microbubbles. Methods Cultured VSMCs were exposed to a low-frequency continuous wave ultrasound （1 MHz, 0.3 W/cm^2 , 120 s） in the presence of liposome microbubbles （1 μl/ml）. Apoptosis of cells were detected by flow cytometry with double staining of fluorescein isothiocyanate （FITC）-labeled Annexin V/propidium iodide （PI）, and the expression of Bax and Bcl-2 were determined by immunocytochemical stain, while intracellular free calcium concentration （[Ca^2＋ ]i ） was detected by laser confocal microscopy with fluorescent Ca^2＋-sensitive probe fura-4/acetoxylmethyl ester （Fura-4/AM）. Results Compared with control group and platelet derived growth factor-BB treatment group （PDGF）, the percentage of apoptosis and the expression of Bax increased markedly in ultrasound combined with microbubbles group （US＋ MB） and ultrasound combined with mierobubbles and PDGF treatment group （PDGF＋ US＋ MB, P〈0.01）, while the expression of Bcl-2 in the two groups decreased significantly （P〈0.01）. Compared with （US＋MB） group, the apoptosis cells, upregulation of expression of Bax and reduction of expression of Bcl-2 in （PDGF＋ US＋ MB） group were more obvious （P〈0.01）. [Ca^2＋ ]i in other tested groups increased significantly, compared with that in the control. [Ca^2＋ ]i in （US＋MB） group and （PDGF＋US＋MB） group were lower than that in PDGF group （P〈0.01）. Conclusion Low frequency ultrasound in combination with microbubbles can induce VSMCs apoptosis, which may be related to increase of [Ca^2＋ ]i, upregulation of expression of Bax and reduction of expression of Bcl-2.