ISSN 0371-0874, CN 31-1352/Q



侯嘉云, 宋东莉, 靳大庆, 胡晶莹, 王向东



有研究显示,组蛋白去乙酰基酶复合物(BRAF-HDAC complex, BHC)组分BHC80基因敲除导致小鼠出生一天内死亡。为此,本研究在斑马鱼中下调BHC80基因表达,以深入研究其在生物体发育中的作用。根据斑马鱼BHC80序列,设计合成吗啡啉修饰的反义寡核苷酸,并将其显微注射到单细胞或双细胞期的野生型胚胎中,并用RT-PCR方法验证其有效性。分析BHC80基因阻抑后对胚胎发育,尤其心脏表型和功能的影响。结果显示,吗啡啉修饰的反义寡核苷酸有效下调BHC80基因表达,其对胚胎发育异常的影响呈剂量依赖性。BHC80基因表达下调的斑马鱼胚胎心脏出现多种异常的表型,包括心房心室大小异常、环化不完全、严重者心脏发育呈管状、心搏减弱,心率减慢、心室收缩分数降低。结果表明,BHC80基因表达下调致使胚胎心脏发育异常,心功能受损,这可能是BHC80基因敲除哺乳动物出生后很快死亡的重要原因之一,为进一步阐明心脏发育机制提供了很好的研究工具。

关键词: 斑马鱼; 组蛋白去乙酰基酶复合物蛋白80; 吗啡啉反义寡核苷酸; 心脏发育


[Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish.] [Article in Chinese]

HOU Jia-Yun, SONG Dong-Li, JIN Da-Qing, HU Jing-Ying, WANG Xiang-Dong

Zhongshan Hospital, Fudan University,Shanghai 200032,China; School of Life Sciences, Fudan University,Shanghai 200433,China; Key Laboratory of Molecular Medicine,Ministry of Education, Fudan University,Shanghai 200032,China


The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.

Key words: zebrafish; BHC80; morpholino antisense oligonucleotides; cardiac development

收稿日期:2013-04-08  录用日期:2013-07-02

通讯作者:王向东  E-mail:


侯嘉云, 宋东莉, 靳大庆, 胡晶莹, 王向东. BHC80基因表达下调对斑马鱼胚胎心脏发育的损伤作用[J]. 生理学报 2013; 65 (5): 547-552.

HOU Jia-Yun, SONG Dong-Li, JIN Da-Qing, HU Jing-Ying, WANG Xiang-Dong. [Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (5): 547-552 (in Chinese with English abstract).