中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (5): 402-407.doi: 10.35541/cjd.20200617

• 论著 • 上一篇    下一篇

nicastrin基因沉默的斑马鱼模型构建及其色素异常机制的初步研究

李文锐1    贾苇雪1    张蕴斌2    林麟1    李诚让1   

  1. 1中国医学科学院、北京协和医学院皮肤病医院皮肤科  江苏省皮肤病与性病分子生物学重点实验室，南京  210042；2中国科学院生物化学与细胞生物学研究所，上海  200031
  • 收稿日期:2020-06-18 修回日期:2021-01-26 发布日期:2021-04-29
  • 通讯作者: 林麟；李诚让 E-mail:dllon3@163.com;nylcr72@163.com
  • 作者简介:作者5月份毕业急用
  • 基金资助:
    国家自然科学基金（81472872）；中国医学科学院医学与健康科技创新工程项目（2016-I2M-1-002）

Construction of a nicastrin gene-silenced zebrafish model and a primary study on the mechanism of abnormal pigmentation

Li Wenrui1, Jia Weixue1, Zhang Yunbin2, Lin Lin1, Li Chengrang1   

  1. 1Department of Dermatology, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China; 2Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
  • Received:2020-06-18 Revised:2021-01-26 Published:2021-04-29
  • Contact: Lin Lin; Li Chengrang E-mail:dllon3@163.com;nylcr72@163.com
  • Supported by:
    National Natural Science Foundation of China （81472872）; CAMS Innovation Fund for Medical Sciences （2016-I2M-1-002）

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摘要： 【摘要】 目的 探讨nicastrin（nct）基因对斑马鱼黑素细胞生物学功能的影响。方法 利用吗啉代寡核苷酸（MO）技术针对斑马鱼nct基因mRNA设计nct-MO序列，同时设计对照MO序列（ctrl-MO），并构建5′端为MO靶序列的增强绿色荧光蛋白（EGFP）mRNA，通过显微注射的方式将nct-MO或ctrl-MO与EGFP mRNA共注射入斑马鱼胚胎中，验证nct-MO的沉默效率，观察其表型变化。以野生型斑马鱼作为空白对照组，实时荧光定量PCR检测黑素合成notch信号通路相关基因mitfa、tyr、tyrp1a、tyrp1b、dct、pmela、notch1a、notch1b、hey1 mRNA表达水平的变化。多组间均数比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果 斑马鱼受精后8 h，ctrl-MO + EGFP mRNA组斑马鱼胚胎中均有绿色荧光表达，而nct-MO + EGFP mRNA组及空白对照组胚胎均未见绿色荧光。受精后48 h，nct-MO组幼鱼尾部色素沉着区面积占比（0.169 ± 0.083）低于ctrl-MO组（0.258 ± 0.042，t = 3.202，P = 0.005），且nct-MO组色素分布紊乱。实时荧光定量PCR显示，nct-MO组、ctrl-MO组、空白对照组间pmela、tyrp1a、hey1 基因mRNA 相对表达量差异有统计学意义（均P < 0.05），mitfa、tyr、tyrp1b、dct、notch1a、notch1b mRNA相对表达差异无统计学意义（均P > 0.05）；nct-MO组pmela、tyrp1a相对表达水平（0.708 ± 0.028、0.558 ± 0.136）低于ctrl-MO组（1.023 ± 0.142、1.016 ± 0.134，均P < 0.05）。结论 nct基因可能通过调控斑马鱼黑素合成影响黑素细胞生物学功能。

关键词: 色素沉着异常, 化脓性汗腺炎, Nicastrin基因, γ分泌酶, 斑马鱼模型

Abstract: 【Abstract】 Objective To evaluate the effect of nicastrin （nct） gene on the biological functions of melanocytes in zebrafish. Methods By using a morpholino oligonucleotide （MO） technology, a nct-MO sequence targeting the zebrafish nct mRNA was designed, so was a MO control （ctrl-MO） sequence. Then, the enhanced green fluorescent protein （EGFP） mRNA with MO target sequence at its 5′ end was synthesized, and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish. With wild-type zebrafish as a blank control group, real-time fluorescence-based quantitative PCR （RT-PCR） was conducted to determine the mRNA expression of melanin synthesis- and notch signaling pathway-related genes, including mitfa, tyr, tyrp1a, tyrp1b, dct, pmela, notch1a, notch1b and hey1 genes. One-way analysis of variance was used for the comparison of means among multiple groups, and least significant difference（LSD）-t test for multiple comparisons. Results Eight hours after zebrafish fertilization, green fluorescence was observed in the zebrafish embryos in the ctrl-MO + EGFP mRNA group, but not in the nct-MO + EGFP mRNA group or blank control group. Forty-eight hours after fertilization, the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group （0.169 ± 0.083） than in the ctrl-MO group （0.258 ± 0.042, t = 3.202, P = 0.005）, and disorderly pigment distribution in the tails was observed in the nct-MO group. RT-PCR revealed significant differences in the mRNA expression of pmela, tyrp1a and hey1 genes among the nct-MO group, ctrl-MO group and blank control group （all P < 0.05）, but no significant difference was observed in the mRNA expression of mitfa, tyr, tyrp1b, dct, notch1a or notch1b genes among the 3 groups （all P＞0.05）; the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group （0.708 ± 0.028, 0.558 ± 0.136, respectively） than in the ctrl-MO group （1.023 ± 0.142, 1.016 ± 0.134, respectively, both P < 0.05）. Conclusion The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.

Key words: Pigmentation disorders, Hidradenitis suppurativa, Nicastrin, γ-Secretase, Zebrafish models