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 epididymal epithelial cell apoptosis and its mechanism in vitro

作　　者：李国停 钟瑞华 周洁芸 谢淑武 陈平[2] 钱越英 朱焰 

机构地区：[1]复旦大学生殖与发育研究院上海市计划生育科学研究所药物发展室生殖药理组国家人口和计划生育委员会计划生育药具重点实验室,上海200032 [2]浙江大学理学院化学系,浙江杭州310028 [3]中国科学院物理化学研究所,北京100190

出　　处：《中国药理学通报》2017年第10期1357-1363,共7页Chinese Pharmacological Bulletin

基　　金："十二五"国家科技支撑计划(No 2012BAI31B08);国家自然科学基金资助项目(No 81100463)

摘　　要：目的探讨鬼臼毒素(podophyllotoxin,PPT)抑制SD大鼠附睾上皮细胞增殖作用,揭示其诱导凋亡的相关分子机制。方法体外培养大鼠原代附睾上皮细胞,CCK-8法检测鬼臼毒素作用24、48、72 h后对细胞增殖的影响;透射电镜观察细胞内部结构变化;Annexin V-FITC/PI检测细胞凋亡率;原位末端标记法(TUNEL)观察细胞凋亡形态;RT-PCR技术研究TNF-αmRNA的变化;ELISA法检测鬼臼毒素对细胞培养上清液TNF-α水平的影响;Western blot法检测细胞色素C、caspase-3、caspase-8、caspase-9蛋白的表达水平。结果CCK-8结果显示,鬼臼毒素能明显抑制大鼠附睾上皮细胞增殖并诱导凋亡,并呈剂量及时间依赖性(P<0.05),24、48、72 h的半数抑制浓度(95%置信区间)分别为59.36(15.50~227.41)、0.37(0.080~1.70)、0.077(0.017~0.35)μmol·L^(-1);透射电镜下观察发现,鬼臼毒素引起附睾上皮细胞形态变圆,细胞核破碎,线粒体空泡化;RT-PCR和ELISA检测结果显示,鬼臼毒素能升高TNF-αmRNA水平及细胞培养上清中TNF-α的含量;Western blot结果显示,鬼臼毒素能明显增加细胞中细胞色素C、caspase-3、caspase-8、caspase-9蛋白表达。结论鬼臼毒素可通过线粒体调控的内源性通路和TNF受体介导的外源性通路,诱导大鼠附睾上皮细胞发生凋亡。AimTo investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.MethodsPrimary 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.Annex-in V-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The Td T-mediated d UTP 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-αin cell culture supernatant was measured by enzyme-linked immunosorbent assay（ELISA）technology.Western blot was performed 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 24,48and 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-αmRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C,caspase-3,caspase-8 and caspase-9.ConclusionPodophyllotoxin 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 

分 类 号：R-33[医药卫生] R284.1