BHC80基因表达下调对斑马鱼胚胎心脏发育的损伤作用

Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish

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芋列,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。

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% 02 are supplied through their skin. Therefore, a model ofBHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonu- eleotides 2 （BHC80-M02） 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-M02 injection groups, including 4 rig, 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：g/＇p transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-M02 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-M02 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of eon-MO group in RT-PCR analysis, and the 5 l bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-M02 dosage. The proper BHC80-M02 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-M02 embryos exhibited abnormal cardiac phenotype, including imbalance ot me proporuon oL nea^t 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-M02 embryos may be one of the probable reasons for the BHCSO gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.