CAJ | 학술논문

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、0.5 W/cm2超声干预组、1.3 W/cm2超声干预组和2.0 W/cm2超声干预组.空白对照组切断电源给予假超声干预,0.5 W/cm2超声干预组、1.3 W/cm2超声干预组和2.0 W/cm2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,0.5 W/cm2超声干预组、1.3 W/cm2超声干预组和2.0 W/cm2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).0.5 W/cm2超声干预组、1.3 W/cm2超声干预组和2.0 W/cm2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DNA ladder,并于6h、9h时出现明显片段化.1.3 W/cm2超声干预组和2.0 W/cm2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.
목적 관찰저강도초성(LIPUS)대간종류세포계SMMC-7721세포조망적체외생물학효응,병탐토기가능적작용궤제.방법 장체외배양적SMMC-7721세포근거유무초성간예혹초성작용강도분위공백대조조、0.5 W/cm2초성간예조、1.3 W/cm2초성간예조화2.0 W/cm2초성간예조.공백대조조절단전원급여가초성간예,0.5 W/cm2초성간예조、1.3 W/cm2초성간예조화2.0 W/cm2초성간예조칙근거대응적강도진행초성간예.4조세포초성간예후부육6h,분별채용류식세포의검측세포조망、배사정황급세포주기개변,채용투사전경관찰미결구변화,채용DNA경지당응효전영실험검측DNA시부발생편단화,채용Western-blot법검측Caspase-3단백표체정황.결과 경LIPUS처리후6h,0.5 W/cm2초성간예조、1.3 W/cm2초성간예조화2.0 W/cm2초성간예조세포조망솔분별위4.66％、8.99％、32.41％;차각조G2기(DNA합성후기)세포소점비례증가,G1기(DNA합성전기)세포현저감소,분별여공백대조조동기비교,차이균유통계학의의(P＜0.05).0.5 W/cm2초성간예조、1.3 W/cm2초성간예조화2.0 W/cm2초성간예조Hoechst-염색화투사전경하균가견명현세포조망,차DNA경지당응효전영가견DNA ladder,병우6h、9h시출현명현편단화.1.3 W/cm2초성간예조화2.0 W/cm2초성간예조Caspase-3적표체명현증고,차여공백대조조비교,차이균유통계학의의(P＜0.05).결론 저강도초성가통과다충효응억제간종류세포SMMC-7721증식,유도기조망,차대종류세포적유도조망작용재일정범위내구유강도의뢰성;촉조망작용적궤제가능여경선립체통로특별시Caspase-3단백적활화유관.
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/cm2,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 microstructural 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/cm2 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.