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。
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:A significantly reduced hemoglobin content in Hb^(+/-)embryos and ectopic in 50 hpf 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.
作者
黄梓熙
孟琳
陈良标
陈忠
HUANG Zixi;MENG Lin;CHEN Liangbiao;CHEN Zhong(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;Department of Cardiology,Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 201306,China)
出处
《南京医科大学学报（自然科学版）》
CAS
北大核心
2023年第7期900-908,共9页
Journal of Nanjing Medical University(Natural Sciences)
基金
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。
