超声联合微泡对不同细胞周期血管平滑肌细胞增殖和凋亡的影响

Effect of microbubbles mediated ultrasound insonation on proliferation and apoptosis of vascular smooth muscle cells in different phases of cell cycle

目的探讨超声联合微泡对不同细胞周期血管平滑肌细胞（VSMCs）增殖和凋亡的影响。方法组织贴块法培养大鼠胸主动脉VSMCs，采用血清饥饿法和胸苷双阻断法使细胞同步化，并用流式细胞仪分析同步化结果。以频率1MHz、声强0．3w／cm^2的连续波超声联合脂质微泡辐照不同细胞周期的VSMCs120S，分别用噻唑蓝（MTT）比色法、流式细胞仪AnnexinV／PI染色、免疫细胞化学技术检测VSMCs的增殖、凋亡和增殖细胞核抗原（PCNA）表达。结果成功获得同步化的G0／G1期和S期VSMCs，其同步化率分别为89．53％和66．87％。超声联合微泡可显著抑制S期细胞的增殖并下调PCNA蛋白表达，对G0／G1期细胞的增殖和PCNA表达无明显影响；G0／G1期和S期细胞的凋亡率均较辐照前增高，且对于S期细胞超声联合微泡诱导凋亡的作用更为显著。结论超声联合微泡对处于增殖状态的VSMCs具有显著的抑制增殖和诱导凋亡的作用。

Objective To investigate the effect of microbubbles mediated ultrasound insonation on proliferation and apoptosis of vascular smooth muscle cells （VSMCs） in different phase of cell cycle. Methods Rat thoracic aortic VSMCs were cultured in vitro by the method of tissue adherence. The cells were synchronized by the methods of serum starvation and double thymidine block. The synchronization results were detected by flow cytometer. VSMCs in different phases of cell cycle were exposed to 1 MHz continuous waves ultrasound for 120 s at intensity 0.3 W/cm2 in the presence of lipid-coated microbubbles （1 ml/L）. Apoptosis of VSMCs was analyzed by AnnexinV/PI staining using flow cytometry. The proliferation and the proliferating cell nuclear antigen（PCNA） protein expression of VSMCs were detected by MTT assay and immunocytochemistry, respectively. Results The synchronized G0/G1 and S phase VSMCs were achieved,with synchronized rates to 89.53% and 66.87% ,respectively. Ultrasound sonication for 120 s with microbubbles could significantly inhibit the proliferation and downregulate the PCNA expression of S phase VSMCs,but the proliferation and PCNA expression of G0/G1 phase VSMCs were not affected. After treatment of ultrasound with microbubbles,the apoptotic ratio were found to reach （7.05± 2.04） % in G0/G1 phase VSMCs and （27.01 ± 3.87）% in S phase VSMCs. Conclusions Microbubbles mediated ultrasound insonation can significantly inhabit the proliferation and induce apoptosis in VSMCs at proliferation stage.