电化学疗法对乳腺癌MCF-7 细胞凋亡及细胞内钙离子浓度的影响

doi:10.3877/cma. j. issn.1674-0807.2015.01.004

目的

探讨电化学疗法(ECT)对乳腺癌MCF-7 细胞内钙离子浓度及细胞凋亡、坏死和细胞生长抑制的影响。

方法

将MCF-7 细胞分为对照组及实验组,对照组为0 C(C: 库仑),实验组按电量不同分为4 组: 3 C、5 C、10 C 及20 C 组。每组重复6 孔,用不同电量处理MCF-7 细胞后培养6 h 和24 h,通过MTT 法、GENMED 细胞钙离子浓度比色法、流式细胞仪和激光共聚焦分别检测细胞生长抑制率、细胞内Ca²⁺浓度、观察凋亡和坏死情况。组间均数比较采用方差分析,应用SNK 方法进行两两比较, 应用5×2 析因分析探讨电量与时间对细胞的影响。

结果

不同电量干预MCF-7 细胞后培养6 h 及24 h,细胞生长抑制率(6 h: F=508.43,P=0.000;24 h: F=232.76, P=0.000)、坏死率(6 h: F=2282.07,P=0.000;24 h: F=2644.60,P=0.000)、细胞内Ca²⁺浓度(6 h: F=1416.06,P=0.000;24 h: F=2394.26,P=0.000)随电量增加而增加,电量及培养时间之间具有交互作用(F=288.93,P=0.000;F=2305.39,P=0.000;F=1651.96,P=0.000)。流式细胞仪检测细胞凋亡率结果显示,细胞凋亡率在3、5、10 C 组明显增高,而20 C 组细胞凋亡率降低,电量及培养时间有交互作用(F=51.20,P=0.000)。

结论

ECT 可抑制乳腺癌MCF-7 细胞株的生长并诱导其凋亡,并引起细胞内Ca²⁺浓度升高。低剂量ECT 主要诱导细胞凋亡,高剂量ECT 主要引起细胞坏死。

关键词: 乳腺肿瘤, 电化学疗法, MCF-7 细胞, 细胞凋亡, 坏死

Objective

To investigate the influence of electrochemical therapy(ECT) on intracellular Ca²⁺ concentration, apoptosis and necrosis, and proliferation inhibition in breast cancer MCF-7 cells.

Methods

MCF-7 cells were randomly divided into 5 groups treated by different quantities of electricity,i.e.,0 c(control group),3 C,5 C,10 C,20 C (experimental groups).We replicated 6 wells in each group.The cells were cultured for 6 h and 24 h after electrochemical treatment.MTT method,GENMED calcium colorimetric assay and flow cytometry assay were used to detect the inhibitory rate of cells growth and intracellular Ca²⁺concentration,respectively.The confocal laser scanning microscope was used to observe the apoptosis and necrosis of MCF-7 cells.The means between the two groups were compared using analysis of variance, SNK method was used for pairwise comparison, and 5 × 2 factorial analysis was performed to explore the effect of quantity of electricity and time on the cells.

Results

At 6 h and 24 h of culture after ECT with different quantities of electricity,the inhibition rate of MCF-7 cells (6h: F=508.43,P=0.000;24 h: F=232.76,P=0.000),necrosis rate (6 h: F=2282.07,P=0.000;24 h: F=2644.6,P=0.000),and intracellular Ca²⁺ concentration (6 h: F=1416.06,P=0.000;24 h: F=2394.26,P=0.000) were significantly increased with the increase of quantity of electricity,and the interaction was observed between quantity of electricity and culture time (F=288.93,P=0.000;F=2305.39,P=0.000;F=1651.96,P=0.000). Flow cytometry showed that the apoptosis rate was significantly increased in 3 C,5 C,10 C groups,and then decreased in 20 C group,the interaction was observed between quantity of electricity and culture time (F=51.20,P=0. 000).

Conclusions

ECT can inhibit the cell growth and induce the apoptosis of MCF-7 cells and increase the concentration of intracellular Ca²⁺.However,low dose of ECT mainly induces cell apoptosis and high dose of ECT mainly causes cell necrosis.

Key words: Breast neoplasms, Electrochemical therapy, MCF-7 cells, Cell apoptosis, Necrosis

表1 不同治疗电量下人乳腺癌MCF-7 细胞的细胞抑制率的变化(%)

组别	抑制率^b
组别	6 h	24 h
0 C	0	0
3 C	16.96±6.07	26.93±11.01
5 C	30.32±9.07	63.53±12.74
10 C	83.14±3.54	90.43±4.35
20 C	94.27±2.02	98.67±0.56
F值^a	508.43	232.76
P值	0.000	0.000

表1 不同治疗电量下人乳腺癌MCF-7 细胞的细胞抑制率的变化(%)

组别	抑制率^b
组别	6 h	24 h
0 C	0	0
3 C	16.96±6.07	26.93±11.01
5 C	30.32±9.07	63.53±12.74
10 C	83.14±3.54	90.43±4.35
20 C	94.27±2.02	98.67±0.56
F值^a	508.43	232.76
P值	0.000	0.000

图1 MCF-7 细胞在激光共聚焦显微镜下的图像注: a 图为6 h 10 C 组(×200);b 图为6 h 20 C 组(×200);c 图为24 h 10 C 组(×200);d 图为24 h 20 C 组(×200);蓝色箭头所示为早期凋亡细胞;红色箭头所示为晚期凋亡细胞;绿色箭头所示为坏死细胞

表2 不同电量及作用时间对人乳腺癌MCF-7 细胞凋亡率和坏死率的影响(%)

组别	凋亡率^b		坏死率^c
组别	6 h	24 h	6 h	24 h
0 C	3.08±0.75	3.29±0.55	6.64±0.54	6.66±0.96
3 C	14.71±0.84	23.32±1.21	10.52±1.54	19.77±1.07
5 C	30.01±1.57	35.34±1.20	15.14±1.56	25.03±1.02
10 C	37.63±1.58	51.08±2.56	18.26±1.19	30.35±1.27
20 C	7.54±1.45	14.92±0.96	63.05±1.96	79.54±2.65
F值^a	1151.53	1272.61	2282.07	2644.60
P值	0.000	0.000	0.000	0.000

表2 不同电量及作用时间对人乳腺癌MCF-7 细胞凋亡率和坏死率的影响(%)

组别	凋亡率^b		坏死率^c
组别	6 h	24 h	6 h	24 h
0 C	3.08±0.75	3.29±0.55	6.64±0.54	6.66±0.96
3 C	14.71±0.84	23.32±1.21	10.52±1.54	19.77±1.07
5 C	30.01±1.57	35.34±1.20	15.14±1.56	25.03±1.02
10 C	37.63±1.58	51.08±2.56	18.26±1.19	30.35±1.27
20 C	7.54±1.45	14.92±0.96	63.05±1.96	79.54±2.65
F值^a	1151.53	1272.61	2282.07	2644.60
P值	0.000	0.000	0.000	0.000

图2 流式细胞仪显示细胞凋亡率的变化注: a 图为6 h 0 C 组;b 图为24 h 0 C 组;c 图为6 h 10 C 组;d 图为24 h 20 C 组

表3 不同电量及作用时间对人乳腺癌MCF-7 细胞内Ca²⁺浓度的影响(mmol/L)

组别	Ca²⁺浓度^b
组别	6 h	24 h
0 C	0.021±0.002	0.022±0.002
3 C	0.130±0.003	0.155±0.007
5 C	0.200±0.024	0.283±0.009
10 C	0.333±0.033	0.480±0.019
20 C	0.843±0.035	0.945±0.041
F值^a	1416.06	2394.26
P值	0.000	0.000

表3 不同电量及作用时间对人乳腺癌MCF-7 细胞内Ca²⁺浓度的影响(mmol/L)

组别	Ca²⁺浓度^b
组别	6 h	24 h
0 C	0.021±0.002	0.022±0.002
3 C	0.130±0.003	0.155±0.007
5 C	0.200±0.024	0.283±0.009
10 C	0.333±0.033	0.480±0.019
20 C	0.843±0.035	0.945±0.041
F值^a	1416.06	2394.26
P值	0.000	0.000

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Influence of electrochemical therapy on the apoptosis and intracellular Ca ² ⁺ concentration in breast cancer MCF-7 cells

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Influence of electrochemical therapy on the apoptosis and intracellular Ca 2 + concentration in breast cancer MCF-7 cells

Influence of electrochemical therapy on the apoptosis and intracellular Ca ² ⁺ concentration in breast cancer MCF-7 cells