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HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响
引用本文:罗军涛,李艳,陶筱帆,刘玮,倪国军,张俊芳,韩兵社.HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响[J].中国水产科学,2017,24(5):988-994.
作者姓名:罗军涛  李艳  陶筱帆  刘玮  倪国军  张俊芳  韩兵社
作者单位:省部共建水产种质资源发掘与利用教育部重点实验室,上海海洋大学水产与生命学院,上海201306
基金项目:国家自然科学基金项目(31372516),上海市人才发展资金项目(201457),上海市教育委员会“东方学者”计划支持项目
摘    要:为研究热休克蛋白90(heat shock protein 90,HSP90)在斑马鱼(Danio rerio)胚胎发育中的作用,本实验采用2μmol/L、5μmol/L、10μmol/L的HSP90抑制剂根赤壳菌素(radicicol)对斑马鱼发育期胚胎进行处理,监测斑马鱼胚胎不同发育时期两个HSP90功能抑制标志基因BAG3(BCL2-associated athanogene3)和HSPB1(heat shock protein beta-1)的mRNA的表达水平,并观察不同发育时期的胚胎发育状况。结果如下:(1)实时荧光定量PCR结果显示,BAG3和HSPB1 mRNA水平在根赤壳菌素处理胚胎12 hpf(hours post-fertilization,hpf)或24 hpf后显著增高,Western blot检测到5μmol/L根赤壳菌素处理胚胎24 hpf后HSP70表达上调,证明该实验条件下HSP90功能受到抑制;(2)根赤壳菌素处理后斑马鱼胚胎发育变缓,胚胎成活率统计显示:2μmol/L、5μmol/L、10μmol/L根赤壳菌素处理24 hpf胚胎成活率分别是95%、77%、35%,成活率随根赤壳菌素浓度的增高而降低;(3)5μmol/L根赤壳菌素处理72 hpf可见部分个体色素沉积减少、心包膜增大、肌肉萎缩等发育畸形。研究结果证明HSP90在斑马鱼胚胎发育过程中发挥重要的作用,根赤壳菌素在5μmol/L时已达到较佳抑制效果且成活率较高,而且胚胎发育出现了各种形态学变化,为后期研究HSP90调节斑马鱼胚胎发育奠定了基础。

关 键 词:斑马鱼  热休克蛋白90  根赤壳菌素  色素沉积  发育
修稿时间:2017/9/12 0:00:00

Effect of HSP90 inhibitor radicicol on zebrafish embryonic development
LUO Juntao,LI Yan,TAO Xiaofan,LIU Wei,NI Guojun,ZHANG Junfang,HAN Bingshe.Effect of HSP90 inhibitor radicicol on zebrafish embryonic development[J].Journal of Fishery Sciences of China,2017,24(5):988-994.
Authors:LUO Juntao  LI Yan  TAO Xiaofan  LIU Wei  NI Guojun  ZHANG Junfang  HAN Bingshe
Institution:Key Laboratory of Aquacultural Resources and Utilization, Ministry of Education;College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China
Abstract:To study the role of heat shock protein 90 (HSP90) in the development of zebrafish embryos,we examined the mRNA levels of two HSP90 activity marker genes,namely bag3 (BCL2-associated athanogene 3) and hspb1 (heat shock protein beta-l),at different developmental stages.Furthermore,we observed the marker gene expression and development of zebrafish embryos at different stages after treating with 2 μmol/L,5 μmol/L,and 10 μmol/L of the HSP90 inhibitor radicicol.Analysis by quantitative polymerase chain reaction showed a strong increase in the mRNA levels of bag3 and hspb1 when zebrafish embryos were treated with radicicol at 12 or 24 hours post-fertilization (hpf).Western blot results showed that the protein level of HSP70 was upregulated when zebrafish embryos were treated with 5 μmol/L radicicol at 24 hpf,indicating that the activity of HSP90 was inhibited.Observations of embryonic development showed that the radicicol-treated embryos had a delayed development and survival rates of 95%,77%,and 35% after treatment with 2 μmol/L,5 μtmol/L,and 10 μmol/L radicicol,respectively.The embryos manifested some abnormalities including reduced pigmentation,an enlarged pericardial membrane,and muscular dystrophy after treatment with 5 μmol/L radicicol at 72 hpf.The results showed that HSP90 plays an important role on the development of zebrafish embryos.The radicicol concentration of 5 μtmol/L appeared optimal,because it strongly inhibited HSP90 without decreasing the survival rate of zebrafish embryos below 75%.Various morphological changes appeared in the radicicol-treated embryos,laying a foundation for the further study of HSP90's functions in zebrafish embryos.
Keywords:zebrafish  HSP90  radicicol  pigmentation  development
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