中国药理学通报2017，Vol.33Issue(10)：1357-1363,7.DOI:10.3969/j.issn.1001-1978.2017.10.007
鬼臼毒素诱导离体大鼠附睾上皮细胞凋亡机制研究
Study on podophyllotoxin induced rat epididymalepithelial cell apoptosis and its mechanism in vitro
摘要
Abstract
Aim To investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.Methods Primary epididymal epithelial cells were cultured in vitro.CCK-8 assay was used to analyze proliferation of epididymal epithelial cells induced by podophyllotoxin on 24, 48 and 72 h.The ultra structural changes of the epididymal epithelial cells were observed by transmission electron microscope.AnnexinV-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The TdTmediated dUTP nick end labeling(TUNEL) technique was applied to observe the morphological changes of apoptotic cells.The expression of tumor necrosis factor alpha(TNF-α) mRNA was investigated by real-time RT-PCR.The level of TNF-α in cell culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA) technology.Western blot was per-formed to determine the protein expression of cytochrome C, caspase-3, caspase-8, caspase-9.Results Podophyllotoxin significantly inhibited the activity of proliferation and induced apoptosis of epididymal epithelial cells in a dose-and time-dependent manner(P<0.05), with a 50％ inhibitory concentration(IC50) value and corresponding 95％ confidence intervals(CI) of 59.36(15.50～227.41), 0.37(0.080～1.70), 0.077(0.017～0.35) μmol·L-1 at 24, 48 and 72 h, respectively.Podophyllotoxin induced cell volume turned round and cell nuclear fragmentation and mitochondrial vacuolation.RT-PCR and ELISA results showed that podophyllotoxin improved the expression of TNF-α mRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C, caspase-3, caspase-8 and caspase-9.Conclusion Podophyllotoxin can induce rat epididymal epithelial cell apoptosis through both the mitochondria-regulated intrinsic pathway and the TNF receptor-mediated extrinsic pathway.关键词
鬼臼毒素/生育力/大鼠附睾上皮细胞/细胞凋亡/线粒体/TNF-α/caspase活化Key words
podophyllotoxin/fertility/rat epididymal epithelial cell/cell apoptosis/mitochondria/TNF-α/caspase activation分类
医药卫生引用本文复制引用
李国停,钟瑞华,周洁芸,谢淑武,陈平,钱越英,朱焰..鬼臼毒素诱导离体大鼠附睾上皮细胞凋亡机制研究[J].中国药理学通报,2017,33(10):1357-1363,7.基金项目
"十二五"国家科技支撑计划(No 2012BAI31B08) （No 2012BAI31B08）
国家自然科学基金资助项目(No 81100463) （No 81100463）
