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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 article explored the mechanism of apoptosis induced by Nereis active protease (NAP) in SPC-A-1 cells. The inhibitory effect of NAP on SPC-A-1 cells was detected by MTT assay. The morphological changes of SPC-A-1 cells were observed by inverted microscope and AO / EB staining. Flow cytometry was used to detect the cell apoptosis rate and cell membrane potential The changes of apoptosis-related proteins in the cells were detected by Western Blotting. NAP inhibited the activity of SPC-A-1 cells in a dose-and time-dependent manner; NAP-induced morphological changes of apoptotic cells were observed. Flow cytometry showed that with the concentration of NAP The early apoptotic rate of SPC-A-1 cells increased from 13.50% to 22.98%, and the percentage of mitochondrial membrane potential decreased from 12.95% to 25.28%. The results of Western Blotting showed that the ratio of Bax / Bcl-2 significantly increased by 6.05-fold compared with that of the control group after treated with 50μg / mL NAP for 24 h, and the levels of Cyt-C, Cleaved-Caspase 9, Cleaved-Caspase 3 and Cleaved- , The relative expression of the control group reached 2.32,3.07,3.68,1.36 times. The mechanism of NAP-induced apoptosis in SPC-A-1 cells may be through the down-regulation of Bcl-2 protein expression and up-regulation of Bax protein expression, which in turn leads to the decrease of mitochondrial membrane potential, the promotion of Cyt-C transfer and the activation of Caspase family The cascade eventually led to apoptosis in SPC-A-1 cells.