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

作　　者：张国梅[1] 杨最素[1] 丁国芳[1,2] 黄芳芳[1] 赵玉勤[1] 

机构地区：[1]浙江海洋学院食品与医药学院,浙江省海洋生物医用制品工程技术研究中心,浙江舟山316022 [2]浙江省海洋水产研究所,浙江舟山316021

出　　处：《现代食品科技》2015年第3期6-11,共6页Modern Food Science and Technology

基　　金：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)

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,50μg/mL NAP作用24h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。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 24h 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/m L NAP for 24h, 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.

关 键 词：海洋药物 沙蚕 蛋白酶 抗肺癌 细胞凋亡 

分 类 号：R285[医药卫生—中药学]