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目的探讨在斑马鱼胚胎发育中外源性视黄酸(RA)是否能逆转乙醇的毒性。方法采用原位杂交方法检测乙醇和RA共同处理、二者单独处理斑马鱼胚胎后nkx2.5基因的表达及DEAB(视黄醛脱氢酶的抑制剂)与乙醇共同处理及单独处理后nkx2.5基因的表达。采用RT-PCR的方法检测gata5、gata6、raldh2基因的表达,并检测raldh2基因敲除后gata5、gata6的表达变化。结果将RA和乙醇共同处理斑马鱼胚胎后,胚胎发育畸形更加明显,在受精后24 h(24 hpf)nkx2.5的原位杂交结果显示双侧心脏原基不能融合成单个原始心管;乙醇单独处理胚胎影响心脏的环化过程,且nkx2.5的表达上调;乙醇与RA共同处理后,gata5、gata6及raldh2的表达较正常对照组轻微上调,但差异无统计学意义,而敲除raldh2基因后,gata5、gata6的表达有显著下调;DEAB处理胚胎后,原始心管融合正常;300 mmol.L-1乙醇处理后,心脏原基不能融合成原始心管,与200 mmol.L-1乙醇及RA共同处理的表型相似。结论RA和乙醇共同处理后,通过影响原始心管融合而加重心脏发育的畸形,这种现象可能是因为乙醇代谢率降低,加重了乙醇的毒性,并不是由于影响了gata5、gata6的表达;乙醇通过影响nkx2.5基因的表达,而使心脏环化发生异常;RA信号能调节gata5和gata6的表达。
Objective To investigate whether exogenous retinoic acid (RA) can reverse the toxicity of ethanol during zebrafish embryo development. Methods The in situ hybridization was used to detect the co-treatment of ethanol with RA. The expression of nkx2.5 gene in both zebrafish embryos and DEAB (inhibitor of retinaldehyde dehydrogenase) were treated with ethanol and nkx2 alone. 5 gene expression. The expression of gata5, gata6 and raldh2 genes was detected by RT-PCR and the expression of gata5 and gata6 was detected after raldh2 knockout. Results The zebrafish embryos were treated with RA and ethanol. The embryo malformations were more obvious. In situ hybridization of nkx2.5 at 24 h after fertilization showed that the bilateral cardiac precursors could not fuse into single original cardiac tubes. Ethanol Cyclization of the heart was affected by embryo treatment alone, and the expression of nkx2.5 was up-regulated. The expression of gata5, gata6 and raldh2 was slightly upregulated in ethanol treated with RA compared with the normal control group, but the difference was not statistically significant The gene expression of gata5 and gata6 was significantly down-regulated. After embryos were treated with DEAB, the original cardio-vascular fusion was normal. After treated with 300 mmol.L-1 ethanol, Phenotypes similar to ethanol and RA co-treatment. Conclusions The co-treatment of RA and ethanol can aggravate cardiac deformity by affecting primary cardiac fusion. This phenomenon may be attributed to the decrease of ethanol metabolic rate and the aggravation of ethanol toxicity, which is not due to the effect on the expression of gata5 and gata6. Ethanol By affecting the expression of nkx2.5 gene, the heart circulates abnormally; RA signal regulates the expression of gata5 and gata6.