Page 14 - 南京医科大学学报自然科学版

南京医科大学学报（自然科学版） 第43卷第7期
·900 · Journal of Nanjing Medical University（Natural Sciences） 2023年7月

·基础研究·

斑马鱼3号染色体血红蛋白基因敲除对心血管发育的影响

黄梓熙 ，孟 琳 ，陈良标 ，陈 忠 2*
1*
1
1
上海海洋大学水产与生命学院，上海 201306；上海交通大学医学院附属第六人民医院心内科，上海 201306
1 2

［摘 要］ 目的：研究斑马鱼3号染色体血红蛋白基因敲除后心血管发育的差异及可能的机制。方法：利用CRISPR/Cas9敲
除斑马鱼3号染色体血红蛋白基因hbba2到hbae3片段，获得杂合突变体斑马鱼（Hb ）。检测Hb 与野生型斑马鱼（WT）受精
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卵的血红蛋白含量及分布，观察血管的发育，统计两者5月龄红细胞数量，并采用成年斑马鱼心脏转录组测序分析，qRT⁃PCR
进行验证。结果：Hb 胚胎血红蛋白含量明显减少，且在受精后50 h的胚胎中异位分布。Hb 腹部主血管与共同基静脉相对
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WT发育迟缓，肠下静脉发育更快，尾部节间血管增生；而Hb 和WT成鱼红细胞数量的差异无统计学意义（P > 0.05）。对成年
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斑马鱼心脏转录组分析发现，血红蛋白水平降低导致nos2b、cybb上调，hif3a下调，并激活了线粒体的代偿保护机制。结论：血
红蛋白基因敲除影响了胚胎时期斑马鱼的血红蛋白水平和血管发育，通过调控基因表达和线粒体的补偿机制可维持成鱼的正
常生理活动。
［关键词］ 血红蛋白；心血管发育；CRISPR/Cas9；hbba2；hif3a
［中图分类号］ Q344 ［文献标志码］ A ［文章编号］ 1007⁃4368（2023）07⁃900⁃09
doi：10.7655/NYDXBNS20230702

Effect of zebrafish hemoglobin gene knockout in chromosome 3 on cardiovascular

development
1
1
1*
HUANG Zixi ，MENG Lin ，CHEN Liangbiao ，CHEN Zhong 2*
1 College of Fisheries and Life Science，Shanghai Ocean University，Shanghai 201306；Department of Cardiology，
2
Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine，Shanghai 201306，
China

［Abstract］ Objective：To investigate the differences of cardiovascular development in zebrafish after knockout of the hemoglobin
gene on chromosome 3 and the underlying mechanism. Methods：We used CRISPR/Cas9 technology to knock out the fragment from
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hbba2 to hbae3 of the zebrafish hemoglobin gene on chromosome 3 to obtain heterozygous zebrafish（Hb ）. The hemoglobin content
and distribution of Hb +/- and wild⁃type zebrafish（WT）fertilized eggs were detected，the development of blood vessels was observed，
and the number of 5⁃month⁃old erythrocytes of both were counted. The transcriptome of the heart of adult zebrafishes were analyzed by
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sequencing and validated by qRT⁃PCR. Results：A significantly reduced hemoglobin content in Hb embryos and ectopic in 50 hpf
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embryos was detected. Compared with WT，Hb had a retarded development of abdominal major vessels and common cardinal veins in
the abdomen，a more rapid development of the subintestinal veins，and hyperplasia of the caudal intersegmental vessels. There was no
significant difference in erythrocytes between Hb +/- and adult WT（P > 0.05）. Transcriptome analysis of the heart of adult zebrafish
revealed that the decreased hemoglobin levels led to the up⁃regulation of nos2b and cybb，down⁃regulation of hif3a，and activation of
compensatory protective mechanisms in mitochondria. Conclusion：Hemoglobin gene knockout affects hemoglobin levels and vascular
development in embryonic zebrafish，and the normal physiological activity of adult fish can be maintained by regulating gene
expression and mitochondrial compensation mechanism.
［Key words］ hemoglobin；cardiovascular development；CRISPR/Cas9；hbba2；hif3a
［J Nanjing Med Univ，2023，43（07）：900⁃908］

［基金项目］ 国家自然科学基金重点项目（32130109）；上海市第六人民医院特色学科项目
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通信作者（Corresponding author），E⁃mail：zhongchen7498@hotmail.com；lbchen@shou.edu.cn