摘要
目的探讨斑马鱼肠道自发绿色荧光的原因及环孢素A(cyclosporine A,CsA)对自发荧光的影响,优化利用转基因(transgenic,Tg)斑马鱼Tg(abcb4:eGFP)筛选肿瘤多重耐药药物的方法。方法选取受精后5天(five day post fertilization,5 dpf)的野生型(wild type,WT)和Tg(abcb4:eGFP)斑马鱼幼鱼,观察其肠道自发荧光;将鲢鱼胆汁注射到WT斑马鱼胚胎后观察荧光情况;检测不同处理浓度和不同处理时间的CsA对WT斑马鱼肠道自发荧光的影响;10μmol/L CsA处理2 h对Tg(abcb4:eGFP)斑马鱼荧光检测的影响;Western blot检测CsA对Tg(abcb4:eGFP)斑马鱼b4型ATP结合盒(ATP-binding cassette b4,Abcb4)和增强型绿色荧光蛋白(enhanced green fluorescence protein,eGFP)表达的影响。结果斑马鱼肠道存在自发荧光,可影响Tg(abcb4:eGFP)斑马鱼的荧光检测准确度;鲢鱼胆汁在斑马鱼胚胎中可激发出绿色荧光;CsA可抑制斑马鱼肠道自发荧光,其中10μmol/L CsA处理2 h可达到最佳抑制效应,且不影响斑马鱼Abcb4和eGFP蛋白表达。结论斑马鱼肠道自发荧光可能为胆红素所致,10μmol/L CsA处理2 h可有效抑制斑马鱼肠道自发荧光,提高Tg(abcb4:eGFP)斑马鱼药物筛选时荧光检测的准确度。
Objective To investigate the cause of intestinal green autofluorescence in zebrafish and the influence of Cyclosporine A(CsA)on autofluorescence and to optimize the tumor multidrug resistance drug screening system based on transgenic(Tg)(abcb4:eGFP)zebrafish.Method Five day post fertilization(5 dpf)wild type(WT)and Tg(abcb4:eGFP)zebrafish were selected to observe intestinal green autofluorescence.Green fluorescence was observed after silver carp bile was injected into WT zebrafish embryos.The effects of CsA on intestinal green autofluorescence of WT zebrafish at different treatment concentrations and different treatment times were detected.The impact of treatment with CsA(10μmol/L)for 2 h on the fluorescence of Tg(abcb4:eGFP)zebrafish was examined.The effects of CsA on the expression of ATP-binding cassette b4(Abcb4)and enhanced green fluorescence protein(eGFP)were detected by Western blot.Results There was green autofluorescence in the intestine of zebrafish,which can affect the fluorescence detection accuracy of Tg(abcb4:eGFP)zebrafish.Silver carp bile that was injected into zebrafish embryos can stimulate green fluorescence.Treatment with CsA(10μmol/L)for 2 h could inhibit intestinal green autofluorescence of zebrafish.Treatment with CsA(10μmol/L)for 2 h could achieve the best inhibitory effect without affecting the expression of Abcb4 and eGFP in Tg(abcb4:eGFP)zebrafish.Conclusion The intestinal green autofluorescence of zebrafish might be caused by bilirubin.Treatment with CsA(10μmol/L)for 2 h can effectively inhibit intestinal green autofluorescence and improve the accuracy of fluorescence detection in Tg(abcb4:eGFP)zebrafish drug screening.
作者
孔鹏
莫大双
赵砚驰
张乙进
杨雁竹
路政
舒莉萍
何志旭
KONG Peng;MO Dashuang;ZHAO Yanchi;ZHANG Yijin;YANG Yanzhu;LU Zheng;SHU Liping;HE Zhixu(Department of Pediatrics,School of Clinical Medicine,Guiyang 550004,Guizhou,China;Department of Children's Emergency,Guizhou Branch of Shanghai Children's Medical Center Shanghai Jiaotong University School of Medicine,Guiyang,550004,Guizhou,China;Department of Immunology,School of Basic Medicine,Guizhou Medical University,Guiyang 550004,Guizhou,China;School of Clinical Laboratory Science,Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Science,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
2025年第3期370-377,共8页
Journal of Guizhou Medical University
基金
国家自然科学基金(32160168)
贵州省科技计划项目(黔科合平台人才-CXTD[2021]002)
贵州省卫生健康委科学技术基金(gzwkj2023-091)。
