低强度超声诱导人肝癌细胞系SMMC-7721细胞凋亡的体外研究  

Apoptosis of SMMC-7721 cells induced by the low-intensity pulsed ultrasound in vitro

作　　者：石明芳[1] 刘邦忠[1] 杨名珍 薛军[1] 刘光华[1] 王平[1] 李蕴[1] 

机构地区：[1]上海市复旦大学附属中山医院康复科,上海200032

出　　处：《中华物理医学与康复杂志》2015年第8期565-570,共6页Chinese Journal of Physical Medicine and Rehabilitation

摘　　要：目的观察低强度超声（LIPUS）对肝肿瘤细胞系SMMC-7721细胞凋亡的体外生物学效应，并探讨其可能的作用机制。方法将体外培养的SMMC-7721细胞根据有无超声干预或超声作用强度分为空白对照组、0．5W／cm^2超声干预组、1．3W／cm^2超声干预组和2．0W／cm^2超声干预组。空白对照组切断电源给予假超声干预，0．5W／cm^2超声干预组、1．3W／cm^2超声干预组和2．0W／cm^2超声干预组则根据对应的强度进行超声干预。4组细胞超声干预后孵育6h，分别采用流式细胞仪检测细胞凋亡、坏死情况及细胞周期改变，采用透射电镜观察微结构变化，采用DNA琼脂糖凝胶电泳实验检测DNA是否发生片段化，采用Western—blot法检测Caspase-3蛋白表达情况。结果经LIPUS处理后6h，0．5W／cm^2超声干预组、1．3W／cm^2超声干预组和2．0W／cm^2超声干预组细胞凋亡率分别为4．66％、8．99％、32．41％；且各组G2期（DNA合成后期）细胞所占比例增加，G1期（DNA合成前期）细胞显著减少，分别与空白对照组同期比较，差异均有统计学意义（P〈0．05）。0．5W／cm^2超声干预组、1．3W／cm^2超声干预组和2．0W／cm^2超声干预组Hoechst-染色和透射电镜下均可见明显细胞凋亡，且DNA琼脂糖凝胶电泳可见DNAladder，并于6h、9h时出现明显片段化。1．3W／cm^2超声干预组和2．0W／cm^2超声干预组Caspase-3的表达明显增高，且与空白对照组比较，差异均有统计学意义（P〈0．05）。结论低强度超声可通过多种效应抑制肝肿瘤细胞SMMC-7721增殖，诱导其凋亡，且对肿瘤细胞的诱导凋亡作用在一定范围内具有强度依赖性；促凋亡作用的机制可能与经线粒体通路特别是Caspase-3蛋白的活化有关。Objective To explore whether the low-intensity pulsed ultrasound （LIPU） could induce apoptosis on SMMC-7721 cells and to explore the underlying mechanism. Methods The SMMC-7721 cells were randomly divided into 4 groups： a blank control group, which was subject to sham exposure to ultrasound, and 3 ultrasound intervention groups exposed to ultrasound at intensities of 0.5, 1.3 and 2.0 W/cm^2 , respectively. Then they were incubated for 6 h. The cell apoptosis, necrosis and changes of cell cycles were measured using the flow cytometry. The transmission electron microscope （TEM） was used to observe mierostructural changes in the cells. The agarose gel electrophoresis （AGE） was used to examine the DNA fragmentation, and Western-blotting was employed to assess the protein expression of caspase-3. Results The average cell apoptosis rate of the 3 intervention groups were 4.66% , 8, 99% and 32.41% , respectively. The percentage of cells in G2 phase increased significantly and those in G1 phase decreased significantly in the 3 intervention groups compared to the blank control group at the same time points. In the intervention groups, significant cell apoptosis was observed under TEM, and DNA ladders was seen in AGE, with DNA fragments appearing obviously when cells were incubated for 6 h and 9 h after ultrasound exposure. In intervention groups subject to 1.3 and 2.0 W/cm^2 ultrasound exposure, the protein expression of caspase-3 was significantly higher than that of the control group. Conclusion LIPU can inhibit the proliferation and induce apoptosis of SMMC-7721 cells with a dose-dependent feature. The possible mechanism underlying the LIPU-induced cell apoptosis might be related to the activation of the mitochondria pathway, and especially the caspase-3 protein.

关 键 词：低强度超声 人肝癌细胞系SMMC-7721 抗癌效应 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]