低强度超声对HeLa肿瘤细胞凋亡的诱导作用及其机制

doi:10.3877/cma.j.issn.2095-1221.2020.05.004

目的

探讨低强度超声在诱导HeLa肿瘤细胞凋亡中的作用及其机制。

方法

将体外培养的HeLa肿瘤细胞根据超声干预强度分为阴性对照组（切断电源后给予假的超声干预）和0.5、1.3、2.0 W/cm²超声干预组，分别通过MTT实验测定细胞活力、存活率，HE染色检测细胞形态，细胞凋亡实验检测细胞凋亡率，流式细胞术测定ROS含量及Western blot实验检测caspase-12、survivin和内参蛋白β-actin的表达情况。多组间比较采用单因素方差分析，各个实验组与对照组比较采用Dunnet-t检验。

结果

与阴性对照组比较，0.5、1.3、2.0 W/ cm²超声干预组的HeLa肿瘤细胞活力[（99.23±1.56）﹪比（80.52±1.72）﹪、（54.31±1.69）﹪（27.75±1.26）﹪]、细胞存活率[（99.91±1.51）﹪比（76.69±1.92）﹪、（52.57±1.63）﹪、（29.81±1.22）﹪]、survivin蛋白表达（51.19±0.21比43.46±0.34、25.28±0.29、18.32±0.3）均降低，ROS含量（11.21±0.45比24.34±1.23、38.26±2.47、52.18±1.56），细胞凋亡率[（4.23±1.21）﹪比（24.16±1.91）﹪、（48.34±1.66）﹪、（70.27±0.98）﹪]、caspase-12蛋白表达（13.05±0.21比20.23±0.19、33.17±0.32、41.52±0.21）均升高，差异具有统计学意义（P均< 0.05）。

结论

低强度超声可能通过诱导HeLa肿瘤细胞中caspase-12蛋白表达增加和survivin蛋白表达的减少而使HeLa肿瘤细胞发生凋亡。

关键词: Caspase-12, Survivin, 低强度超声, HeLa细胞, 细胞凋亡, 子宫颈癌

objective

To investigate the role and mechanism of low intensity ultrasound induced apoptosis of HeLa tumor cells.

Methods

According to the intensity of ultrasound intervention, HeLa tumor cells cultured in vitro were divided into blank control group (pseudo-ultrasound after power cut off) and setting up three ultrasound intervention groups (0.5, 1.3, 2.0 W/ cm², respectively) . Cell viability and survival rate were determined by MTT, and cell morphology was detected by HE staining. Apoptotic rate was detected by apoptosis experiment. ROS content was determined by flow cytometry and the expression of caspase-12, survivin and the internal reference protein β-actin was detected by Western blot. One-way ANOVA was used for comparison among multiple groups, and Dunnet-t test was used for multiple comparisons. Compared with blank control group, the cell viability (99.23﹪±1.56﹪vs 80.52﹪±1.72﹪, 54.31﹪±1.69﹪, 27.75﹪±1.26﹪, P < 0.05) , cell survival rates (99.91﹪±1.51﹪vs 76.69﹪±1.92﹪, 52.57﹪±1.63﹪, 29.81﹪±1.22﹪, P < 0.05) , ROS content (11.21±0.45 vs 24.34±1.23, 38.26±2.47, 52.18±1.56, P < 0.05) as well as the expression of surviving protein (51.19±0.21 vs 43.46±0.34, 25.28±0.29, 18.32±0.3, P < 0.05) were decreased in ultrasound intervention groups (0.5, 1.3, 2.0 W/cm², respectively) . While the apoptosis rate (4.23﹪±1.21﹪vs 24.16﹪±1.91﹪, 48.34﹪±1.66﹪, 70.27﹪±0.98﹪, P < 0.05) and the expression of caspase-12 protein (13.05±0.21 vs 20.23±0.19, 33.17±0.32, 41.52±0.21, P < 0.05) were increased. The difference was statistically significant (all P < 0.05) .

Conclusion

Low intensity ultrasound may induce the increase of caspase-12 protein expression and decrease of survivin protein expression in HeLa tumor cells, thus leading to apoptosis of HeLa tumor cells.

Key words: Caspase-12, Survivin, Low intensity ultrasound, HeLa cells, Apoptosis, Cervical cancer

表1 Hela肿瘤细胞活力、细胞存活率及凋亡率比较（﹪， ± s）

分组	实验重复次数	细胞活力	细胞存活率	细胞凋亡率
阴性对照组	3	99.23±1.56	99.91±1.51	4.23±1.21
0.5 W/cm²超声干预组	3	80.52±1.72^a	76.69±1.92^a	24.16±1.91^a
1.3 W/cm²超声干预组	3	54.31±1.69^a	52.57±1.63^a	48.34±1.66^a
2.0 W/cm²超声干预组	3	27.75±1.26^a	29.81±1.22^a	70.27±0.98^a
F值		1185.004	1087.433	1121.252
P值		< 0.001	< 0.001	< 0.001

表1 Hela肿瘤细胞活力、细胞存活率及凋亡率比较（﹪， ± s）

分组	实验重复次数	细胞活力	细胞存活率	细胞凋亡率
阴性对照组	3	99.23±1.56	99.91±1.51	4.23±1.21
0.5 W/cm²超声干预组	3	80.52±1.72^a	76.69±1.92^a	24.16±1.91^a
1.3 W/cm²超声干预组	3	54.31±1.69^a	52.57±1.63^a	48.34±1.66^a
2.0 W/cm²超声干预组	3	27.75±1.26^a	29.81±1.22^a	70.27±0.98^a
F值		1185.004	1087.433	1121.252
P值		< 0.001	< 0.001	< 0.001

图1 光学显微镜下观察超声干预对细胞形态的影响（苏木精-伊红染色，×40）

图2 透射电镜下观察超声干预对Hela肿瘤细胞结构（×600）

图3 低强度超声干预对Hela肿瘤细胞中caspase-12、survivin蛋白表达的影响

表2 不同超声强度干预对Hela肿瘤细胞中caspase-12蛋白和survivin蛋白表达水平的影响（ ± s）

分组	实验重复次数	ROS含量	caspase-12蛋白	survivin蛋白
阴性对照组	3	11.21±0.45	13.05±0.21	51.19±0.21
0.5 W/cm²超声干预组	3	24.34±1.23^a	20.23±0.19^a	43.46±0.34^a
1.3 W/cm²超声干预组	3	38.26±2.47^a	33.17±0.32^a	25.28±0.29^a
2.0 W/cm²超声干预组	3	52.18±1.56^a	41.52±0.21^a	18.32±0.31^a
F值		365.400	8634.002	8034.149
P值		< 0.001	< 0.001	< 0.001

表2 不同超声强度干预对Hela肿瘤细胞中caspase-12蛋白和survivin蛋白表达水平的影响（ ± s）

分组	实验重复次数	ROS含量	caspase-12蛋白	survivin蛋白
阴性对照组	3	11.21±0.45	13.05±0.21	51.19±0.21
0.5 W/cm²超声干预组	3	24.34±1.23^a	20.23±0.19^a	43.46±0.34^a
1.3 W/cm²超声干预组	3	38.26±2.47^a	33.17±0.32^a	25.28±0.29^a
2.0 W/cm²超声干预组	3	52.18±1.56^a	41.52±0.21^a	18.32±0.31^a
F值		365.400	8634.002	8034.149
P值		< 0.001	< 0.001	< 0.001

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Effects and mechanisms of low intensity ultrasound-induced apoptosis of HeLa tumor cells

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Effects and mechanisms of low intensity ultrasound-induced apoptosis of HeLa tumor cells