摘要
以TM4细胞（小鼠睾丸支持细胞株）为材料，用不同浓度的玉米赤酶烯酮（zearalenone，ZEA）染毒，利用透射电子显微镜（设0，5，10，20μmol／LZEA浓度组），流式细胞仪、Western blot等技术检测了细胞超微结构、凋亡率、凋亡相关调控蛋白以及内质网应激相关蛋白的变化（设0，0．1，1，10，20μmol／L ZEA浓度组）；通过转染PERK（蛋白激酶R样内质网激酶）siRNA，沉默PERK基因后检测了ZEA诱导TM4细胞凋亡率和凋亡相关蛋白的变化。结果显示：与对照组相比，20μmol／L浓度组损伤最为严重。随着ZEA浓度的增加，细胞凋亡相关蛋白Bax／Bcl-2的值逐渐升高，cleaved caspase 9、cleaved canpase 3蛋白表达量均呈升高趋势，1μmol／L以上染毒组均较各自对照组有极显著差异（P〈0．01）；内质网应激相关蛋白BIP、CHOP的表达量也呈升高趋势，10gmol／L以上染毒组BIP、0．1μmol／L以上染毒组CHOP的表达量均较对照组有显著或极显著差异（P〈0．05或P〈0．01）；PERK通路蛋白含量在10〉mol／L时最高，20，30〉mol／L时逐渐降低，但各染毒组与对照组相比呈显著或极显著差异（P〈0．05或P〈0．01）；TM4细胞caspase3的活性和凋亡率均呈升高趋势，10μmol／L以上染毒组caspase3的活性较对照组均有极显著差异（P〈0．01），各染毒组细胞的凋亡率较对照组均有极显著差异（P〈0．01）。与10μmol／L ZEA处理组相比，siRNA PERK＋10μmol／L ZEA组细胞凋亡率呈显著下降（P〈0．01），Bax／Bcl-2比值、cleaved caspase 3、cleaved caspase 9表达量均下降（P〈0．05或P〈0．01）。结果表明，ZEA可触发TM4细胞内质网应激，通过PERK-eLF2α-ATF4信号通路诱导细胞发生凋亡，PERK信号通路在ZEA诱导TM4细胞凋亡中发挥重要作用。
TM4 cell （Sertoli cells） was choose as test materials, then were infected with different concentration of ZEA. The cell ultrastrueture, apoptosis and apoptosis related proteins and the change of endoplasmic reticulum stress related protein （0,0. 1,1,10,20 μmol/L ZEA concentra- tion group） were detected by transmission electron microscopy （0,5,10,20 μmol/L ZEA concen- tration group） ,flow cytometry and Western blot technology. Changes of apoptosis rate and apop- tosis related proteins in TM4 cells induced by ZEA were detected by transfection of PERK（protein kinase R-like endoplasmic reticulum kinase） siRNA. Compared to the control group, the ultra- structural damage of cells in 20 μmol/L concentration group was the most serious. With the increase of ZEA concentration,apoptosis related protein Bax/Bcl-2 increased gradually,protein expression of cleaved caspase 9,cleaved caspase 3 were in the rising trends（P〈0.01）. Compared to the control group, the expression of endoplasmic reticulum stress related protein BIP and CHOP in the exposure group BIP above 10 μmol/L,CHOP exposure group above 0.1 μmol/L had a signifi- cant or extremely significant difference （P〈0.05 or P〈0.01）. The content of PERK pathway protein was the highest at 10 μmol/L,and decreased gradually at 20 and 30 μmol/L,but the difference between the exposed group and the control group were significant or extremely significant （P〈0.05 or P〈0.01）. Activity and apoptosis rate of TM4 cell Caspase3 were in the increased trends. Compared to the control group, caspase3 activity in the exposure group above 10 μmol/L concentration were significantly （P〈0.01）, compared to the control group, apoptosis rate in all groups of cells were significantly different （P〈0.01）. Compared to 10 μmol/L ZEA treatment group,the apoptosis rate of siRNA PERK＋ 10 μmol/L ZEA group was significantly decreased （P〈0.01） ,Bax/Bcl-2 ratio,expression of the cleaved caspase,cleaved caspase were all decreased （P〈0.05 or P〈0.01）, The results showed that ZEA could trigger endoplasmic reticulum stress in TM4 cells and induce apoptosis through PERK signaling pathway play the important role in ZEA eLF2α-ATF4 signaling pathway,and the PERK induced apoptosis to TM4 cells.
作者
冯楠楠
王冰洁
郑王龙
司梦雪
邹辉
顾建红
袁燕
刘学忠
刘宗平
卞建春
FENG Nan-nan;WANG Bing-jie;ZHENG Wang-long;SI Meng-xue;ZOU Hui;GU Jian-hong;YUAN Yan;LIU Xue-zhong;LIU Zong-ping;BIAN Jian-chun(College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009,China;Jiangsu Co innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第7期1416-1423,共8页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2016YFD0501208)
江苏高校优势学科建设工程资助项目(PAPD)
