目的 研究龙葵碱对人乳腺癌MCF-7细胞凋亡的影响并探讨相关机制。方法 采用四甲基偶氮唑蓝(MTT)法观察不同浓度龙葵碱对人乳腺癌MCF-7细胞的生长抑制作用;4，6-二脒基-2-苯基吲哚(DAPI)染色荧光显微镜进行细胞凋亡核形态学观察;DNA琼脂糖凝胶电泳进行DNA片段化分析，FITC- Annexin V/PI荧光标记流式细胞术检测细胞凋亡率，荧光显微镜结合Fluo-8/Am法、流式细胞术和分光光度比色法分别检测胞内Ca2+浓度、线粒体膜电位(△ψm)和caspase-3，caspase-8活性变化。结果 四甲基偶氮唑蓝法结果显示，龙葵碱对人乳腺癌MCF-7细胞有生长抑制作用。10.0 mmol·L-1龙葵碱处理2 d，4，6-二脒基-2-苯基吲哚染色可见核浓缩及边缘现象，DNA电泳出现特征性的凋亡条带。10.0 mmol·L-1龙葵碱处理1，2，3 d的细胞凋亡率分别为(20.9±7.3)%、(42.6±8.8)% 和(74.9±12.8)%;细胞内Ca2+荧光强度分别为35.6±2.9、52.3±5.6和27.2±2.2;线粒体膜电位(△ψm)值分别下降7.7%、33.2%和46.9%;caspase-8活性在1 d达最高(1.85±0.09)U·μg -1，caspase-3活性则在2 d达最高(2.18±0.09)U·μg -1，与对照组相比均有统计学显著性差异(P＜0.05)。结论 龙葵碱可诱导人乳腺癌MCF-7细胞凋亡，其诱导凋亡的机制可能与胞内Ca2+浓度升高、线粒体膜电位降低和caspase-3、8活化有关。
Abstract
OBJECTIVE To study the effects of solanine on apoptosis of MCF-7 cells and explore the related mechanism. METHODS MTT assay was used to test the inhibitory effect on MCF-7 cellular proliferation with varying concentrations of solanine. The morphological changes of MCF-7 cells were observed with fluorescence microscope after DAPI staining. The analysis of DNA fragmentation on MCF-7 cells was tested by DNA agarose gel electrophoresis. The apoptosis rates and alternation of mitochondrial membrane potential (△ψm) in MCF-7 cells were measured by flow cytometry using fluorescein labeling FITC-Annexin V/PI and JC-1. Intracellular Ca2+ was determined by fluorescence microscope. The activities of caspase-3 and -8 in apoptosis were detected by colorimetric assay. RESULTS The MTT assay showed that solanine inhibited MCF-7 cells proliferation. After 10.0 mmol·L-1 solanine treatment for 2 day, the phenomenon of nuclear condensation and marginalization were shown by DAPI staining in MCF-7 cells, and there was characteristic DNA bands of apoptosis detected by the DNA agarose gel electrophoresis. The apoptosis rate were (20.9±7.3)%、(42.6±8.8)% and (74.9±12.8)% at 1, 2 and 3 d treatment, respectively. The fluorescence intensity of intracellular Ca2+ were 35.6±2.9, 52.3±5.6 and 27.2±2.2 at the same time. The alternation of mitochondrial membrane(△ψm)was decreased to 7.7%, 33.2% and 46.9% compared to control. The maximal activities of intracellular caspase-8 was (1.85±0.09) U·μg -1 after 1 d solanine treatment while the caspase-8 activity reached the highest point after 2 d treatment which was (2.18±0.09) U·μg-1, respectively. There were significantly difference compared with the control groups (P<0.05). CONCLUSION Solanine can inhibit MCF-7 proliferation and induce apoptosis. The molecular mechanism may be related to the increased concentration of intracellular Ca2+, the decrease of mitochondrial membrane potential and the activation of caspase-3,-8.
关键词
龙葵碱 /
MCF-7细胞 /
细胞凋亡 /
钙离子 /
线粒体膜电位 /
半胱氨酸天冬氨酸酶家族
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Key words
solanine /
MCF-7 cell lines /
apoptosis /
Ca2+ /
mitochondrial membrane potential /
caspases
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中图分类号：
R965
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参考文献
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脚注
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基金
嘉兴学院实验技术专项(70113004BW)
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