斑马鱼3号染色体血红蛋白基因敲除影响心血管发育的初步研究
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1.上海海洋大学;2.上海交通大学医学院附属第六人民医院

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]耐寒河鲀品种的耐寒主效基因挖掘和应用研究制研究,国家自然科学基金项目(重点项目)


Effect of zebrafish hemoglobin gene knockout in chromosome 3 on cardiovascular development: a preliminary studyHuang Zixi1, Meng Lin1,Chen Zhong2, Chen Liangbiao1
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    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,并对成年斑马鱼心脏转录组测序分析,qRT-PCR验证。结果:Hb+/-胚胎血红蛋白含量明显减少,且在50 hpf的胚胎中异位分布。Hb+/-腹部主血管与共同基静脉相对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成鱼红细胞数量没有显著差异(P>0.05)。对成年斑马鱼心脏转录组分析发现,血红蛋白水平降低导致NOX2、NOS2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。了解血红蛋白生成在胚胎发生和成年期的调控对于开发新的贫血及心血管疾病疗法非常重要。

    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 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 sequencing and validated by qRT-PCR. Results: Significantly reduced hemoglobin content in Hb+/-embryos and ectopic in 50 hpf embryos were detected. Compared to WT, Hb+/- has retarded development of abdominal major vessels and common cardinal veins in the abdomen, 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 decreased hemoglobin levels led to up-regulation of NOX2 and NOS2, 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. Understanding the regulation of hemoglobin production during embryogenesis and adulthood is important for the development of new therapies for anemia and cardiovascular disease.

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  • 收稿日期:2023-02-20
  • 最后修改日期:2023-03-28
  • 录用日期:2023-07-13
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