ZEA诱导猪卵巢颗粒细胞凋亡机理的研究

【作者】 朱丽； 袁莉芸； 邬静； 李荣芳； 文利新； 袁慧；

【机构】 湖南农业大学动物医学院；

【摘要】 研究目的:以体外培养的猪卵巢颗粒细胞为模型,通过研究ZEA对其毒性作用,来揭示ZEA对猪生殖系统的毒性作用机理;研究方法:采用机械分离法从1-5mm的卵泡中提取猪卵巢颗粒细胞,Giemsa和结晶紫染色观察细胞形态,免疫细胞化学染色方法检测FSHR表达来鉴定颗粒细胞,MTT方法测定细胞生长曲线;于细胞对数生长期加入60,90和120μM的ZEA作用24h后,MTT法检测细胞增殖率,氮蓝四唑(NBT)显色法检测SOD活性,硫代巴比妥酸法测定细胞中MDA的含量,间接法检测细胞中GSH-Px活力,TUNEL和Annexin-FITC/PI双染法检测细胞凋亡,分别用荧光探针DCFH-DA和罗丹明123进行活性氧和线粒体膜电位的检测,western blot检测Caspase-3,9蛋白含量,细胞免疫荧光方法测定Caspase-3蛋白的激活与定位以及Z-VAD-FMK及Ac-LEHD-FMK对细胞凋亡的抑制作用;结果:分离提取的猪卵巢颗粒细胞存活率为65%左右,细胞纯度>97%,且细胞生长状况良好,24h-96h为细胞的对数生长期。高剂量ZEA能抑制猪卵巢颗粒细胞增殖,其IC50为90μM;60μM、90μM和120μM ZEA均极显著降低抗氧化酶SOD、GSH-Px活性,升高MDA的含量而引起细胞氧化损伤,呈剂量效应相关性:并呈剂量依赖性诱导细胞凋亡;研究揭示ZEA通过caspase依赖性的线粒体凋亡途径诱导猪卵巢颗粒细胞凋亡,主要表现在:极显著升高细胞内ROS水平,显著性降低线粒体膜电位,激活Caspase-3,9蛋白并诱导Caspase-3从细胞质进入细胞核执行功能,以及caspase抑制剂Z-VAD-FMK与caspase-9抑制剂Ac-LEHD-FMK显著性抑制ZEA所诱导的猪卵巢颗粒细胞凋亡:结论:ZEA抑制猪卵巢颗粒细胞增殖,引起细胞氧化损伤,并经由caspase依赖性的线粒体凋亡途径诱导细胞凋亡。更多还原

【Abstract】 Objective:The aim of the study was to reveal the toxic effects of the porcine reproductive system through researching the toxic effects of ZEA on the porcine granulosa cells(GCs) in vitro.and to provide a theoretical basis for the detoxification method;Methods:GCs was extracted from follicles(1-5mm) by Mechanical separation.Morphology of GCs were observed by using Giemsa and crytal violet staining,cell viability was determined by 0.4%trypan blue,Immunocytochemistry was used to measure the FSHR expression to characterize GCs.Growth curve of GCs was detected by MIT assay;cell proliferation was determined using MTT method,SOD activity,GSH-Px activity and MDA content were measured by NBT chromogenic assay.TBA assay.and indirect method,respectively.TUNEL and Annexin-FITC/PI staining assay were used to detect apoposis. Measurement of ROS level and mitochondrial transmembrane potential by DCFA and Rh123 staining.Western blotting analysis of caspase-3 and caspase-9 expression,Immunofluorescence for caspase-3 expression and positioning and the inhibition of Z-VAD-FMK and Ac-LEHD-FMK on cell apoptosis:Results:GCs viability was about 65%,purity >97%,and cultured GCs grew well,logarithmic growth phase of GCs was from 24h to 96h. High doses of ZEA inhibits proliferation of porcine GCs,the IC50 was 90μM;60μM,90μM and 120μM ZEA caused oxidative damage through significantly decreased antioxidant enzymes SOD,GSH-Px activity,increased MDA content in a dose-dependent manner.and induced cell apoptosis in a dose-dependent manner.Study reveals ZEA induced apoptosis in porcine ovarian granulosa cells by caspase-dependent mitochondrial apoptotic pathway, mainly in:a significant increase of intracellular ROS levels,significantly decrease of mitochondria]membrane potential,activation of Caspase-3,9 protein and an accumulation and translocation of caspase-3 from the cytoplasm into the nucleus,and the caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Ac-LEHD-FMK significantly inhibited ZEA induced apoptosis in porcine ovarian granulosa cells;Conclusion:ZEA inhibited proliferation of porcine GCs,caused cell oxidative damage and induced apoptosis via the mitochondrial caspase-dependent pathway induced apoptosis.更多还原