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BHC80基因表达下调对斑马鱼胚胎心脏发育的损伤作用

侯嘉云, 宋东莉, 靳大庆, 胡晶莹, 王向东

复旦大学附属中山医院，上海200032；复旦大学生命科学学院，上海200433；复旦大学分子医学教育部重点实验室，上海200032

摘要

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

分类号：R332

[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

Abstract

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: xiangdong.wang@clintransmed.org

引用本文：

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).