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中国应用生理学杂志 ›› 2020, Vol. 36 ›› Issue (6): 662-667.doi: 10.12047/j.cjap.6015.2020.138

• 技术方法 • 上一篇    下一篇

纳米卟啉金属有机骨架对斑马鱼神经系统发育的作用*

王亚洁1, 袁博1,2, 刘伟3, 李玉皓2△, 苑晓勇1△   

  1. 1.天津市眼科医院, 天津 300020;
    2.南开大学医学院, 天津 300071;
    3.天津大学理学院, 天津 300072
  • 收稿日期:2020-02-19 修回日期:2020-11-25 发布日期:2021-03-15
  • 通讯作者: Tel: 022-2731 3336; E-mail: xiaoyong_yuan@hotmail.com
  • 基金资助:
    *国家自然科学基金项目(81970772,81670817,81671179,81971739)

The effect of nanoscale zirconium-porphyrin metal-organic framework on zebrafish embryonic neurodevelopment

WANG Ya-jie1, YUAN Bo1,2, LIU Wei3, LI Yu-hao2△, YUAN Xiao-yong1△   

  1. 1. Tianjin Eye Hospital, Tianjin 300020;
    2. Nankai University School of Medicine, Tianjin 300071;
    3. Tianjin University School of Science, Tianjin 300072, China
  • Received:2020-02-19 Revised:2020-11-25 Published:2021-03-15

摘要: 目的:研究纳米卟啉金属有机骨架(NPMOF)对斑马鱼幼鱼神经系统发育的作用。方法:斑马鱼胚胎在发育6 h(hpf)后随机分为两组:对照组(n=500)和暴露组(n=500),对照组斑马鱼孵化于E3溶液中,暴露组于100 mg/L的NPMOF-E3溶液中,持续暴露至28、48、72、96或120 hpf。以20条斑马鱼为一批,在28、48、72、96和120 hpf分别提取3批实验组和对照组的总RNA用于RT-PCR;在120 hpf,实验组和对照组分别取20条PTU处理过的斑马鱼用于整体胚胎原位杂交,同样是在120 hpf,每组取150条用于免疫荧光染色,30条用于行为学测试。NPMOF的形状和尺寸用透射电镜测定,其光学特性由紫外分光光度计和荧光光谱仪测定;通过免疫荧光、整体胚胎原位杂交和RT-PCR方法检测各类神经细胞的发育;行为学测试用来监测斑马鱼运动状态的改变。结果:与正常组比较,NPMOF暴露组中神经发育相关基因的表达明显升高(P<0.05),müller细胞和星形胶质细胞的数量明显增加(P<0.05),神经元和少突胶质细胞的分布与形态未显示出差异;行为学测试中,斑马鱼的总运动距离、快速运动时间和运动速度的值显著增加(P<0.05),总静止时间明显降低(P<0.05)。结论:100 mg/L NPMOF溶液的持续暴露对斑马鱼神经系统发育,特别是视网膜中müller细胞和脑中星形胶质细胞的发育有促进作用。

关键词: 纳米卟啉金属有机骨架, 神经发育, 中枢神经系统, 视网膜, 斑马鱼

Abstract: Objective: To investigate the effect of nanoscale zirconium-porphyrin metal-organic framework (NPMOF) on the development of nervous system in larval zebrafish. Methods: Embryos of zebrafish were incubated to E3 medium (n=500) or 100 mg/L NPMOF-E3 medium (n=500) from 6 hours post fertilization (hpf) to 28, 48, 72, 96 or 120 hpf. At 28, 48, 72, 96 and 120 hpf, 60 fish were collected respectively for quantitative real-time PCR in both groups. At 120 hpf, 20 fish were used for in situ hybridization, 150 fish were used for immunofluorescence and 30 fish were used for behavioral test, respectively. The shape and size of NPMOF was measured by TEM, and the optical properties were detected by UV-Vis and Fluorescence Spectrometer. In vivo development of multiple neurocytes was examined via in situ hybridization, immunofluorescence and quantitative real-time PCR. Behavioral test was used to manifest the locomotor changes of larval zebrafish. Results: Compared to control group, the mRNA expression levels of neurodevelopment-relative, neuron-relative and neuroglia-relative genes were partially increased obviously after NPMOF exposure (P< 0.05). The distribution and phenotype of neurons and oligodendrocytes showed no significant differences between exposed and unexposed groups, while exposed group showed an increase in the number of müller glia and astrocytes (P<0.05). In behavioral test, there was an increase in total movement distance, fast movement time and velocity and a decrease in total rest time following NPMOF exposure (P<0.05). Conclusion: The data indicate the potential facilitating effect of 100 mg/L NPMOF on neurodevelopment in vivo, especially on the growth of müller glia and astrocytes.

Key words: nanoscale zirconium-porphyrin metal-organic framework, neurodevelopment, central nervous system, retina, zebrafish

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