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首页 > 过刊浏览>2015年第31卷第3期 >2015,31(3):6-11. DOI:10.13982/j.mfst.1673-9078.2015.3.002
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沙蚕活性蛋白酶诱导人肺癌SPC-A-1细胞凋亡的机制研究

The Mechanism of Nereis Active Protease-induced Apoptosis in Lung Cancer SPC-A-1 Cells

发布日期：2015-03-20
作者简介：作者简介：张国梅（1989-），女，硕士生，海洋药物、海洋功能食品；通讯作者：丁国芳
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    [关键词]
    [摘要]
    本文探讨了沙蚕活性蛋白酶（Nereis active protease, NAP）诱导SPC-A-1细胞凋亡机制。采用MTT法检测NAP对SPC-A-1细胞的抑制作用，倒置显微镜及AO/EB染色观察SPC-A-1细胞的形态学的变化，采用流式细胞术检测细胞早期凋亡率和细胞膜电位的变化；并通过Western Blotting检测细胞中凋亡相关蛋白的表达变化。NAP对SPC-A-1细胞活性具有明显的抑制作用且呈现剂量和时间的依赖性；NAP作用后细胞出现凋亡的形态学特征；经流式细胞术检测结果显示，随着NAP作用浓度的增加，SPC-A-1细胞的早期凋亡率从13.50%提高到22.98%，且线粒体膜电位下降所占的百分率从12.95%提高到25.28%。Western Blotting结果显示，50 μg/mL NAP作用24 h后，Bax/Bcl-2的比率相对对照组明显增加了6.05倍；Cyt-C、Cleaved-Caspase 9、Cleaved-Caspase 3、Cleaved-PARP等含量显著上调，相对表达量分别达到对照组的2.32、3.07、3.68、1.36倍。NAP诱导SPC-A-1细胞凋亡的作用机理有可能是通过下调Bcl-2蛋白的表达、上调Bax蛋白的表达，进而诱导线粒体膜电位的下降，促使Cyt-C的转移以及激发Caspase家族发生级联反应最终导致SPC-A-1细胞的凋亡。
    [Key word]
    [Abstract]
    This study aims to investigate the mechanism of Nereis active protease (NAP)-induced SPC-A-1 cell apoptosis. The effect of NAP on the activity of SPC-A-1 cells was examined by MTT assay. Typical morphologic changes were observed in the SPC-A-1 cells by inverted microscopy and AO/EB staining. The early-stage apoptotic rate and membrane potential were detected using flow cytometry, and the protein expressions of apoptosis-related genes were examined via western blotting. The results showed that NAP significantly inhibited the activity of SPC-A-1 cells in a time- and dose-dependent manner. The flow cytometry (FCM) studies revealed that the percentage of the early-stage apoptotic SPC-A-1 cells that were treated with increasing concentrations of NAP for 24 h increased from 13.50% to 22.98%, and the percentage of cells with decreased mitochondrial membrane potential increased from 12.59% to 25.28%. After treatment with 50 μg/mL NAP for 24 h, the Bcl-2/Bax expression ratio relative to the control group increased by a factor of 6.05. The expression levels of Cyt-C, Cleaved-caspase 9, Cleaved-caspase 3, and Cleaved-PARP relative to the control group significantly increased by a factor of 2.32, 3.07, 3.68, and 1.36, respectively. In conclusion, NAP inducing apoptosis of SPC-A-1 cells by decreasing the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of Bax, thereby inducing the decrease of mitochondrial membrane potential which then promoting the transfer of Cyt-C and activate the cascade of caspase family.
    [基金项目]
    国家自然科学基金面上项目（81273429）；浙江省科技厅重大专项（2011C02003，2010R50029，2013C03036）；浙江省自然科学基金立项（LY12C20005，LY12C20008)；舟山市科技计划项目（2012C21013，2012C23023）；
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