CAJ | 학술논문

目的利用新型模式生物斑马鱼,采用外源性视黄醛脱氢酶2抑制剂--对二乙氨基苯甲醛(4-diethylaminobenzaldehyde,DEAB),建立维甲酸(retinoic acid,RA)缺乏的斑马鱼模型,探讨其对斑马鱼胚胎心脏发育的影响. 方法在斑马鱼胚胎受精后5、8、10.3 h,分别用1×10~(-6),5×10~(-6)、10×10~(-6),25×10~(-6) mol/L的DEAB处理,在解剖显微镜下实时观察胚胎发育的全过程,在受精后5 h给予1×10~(-9) mol/L外源性RA干预,观察其对DEAB致畸的拮抗作用.通过胚胎心脏表型观察、心率和心室收缩分数比较以及心脏特异分子标记--心房利钠肽A基因整体原位杂交实验分析RA缺乏对胚胎心脏发育的影响. 结果外源性DEAB处理后,胚胎生存率随着处理浓度增加而降低,随着处理时间点后移而升高.当DEAB浓度≥5×10~(-6) mol/L时,斑马鱼畸胎率为100%,异常表型一致,并能被1×10~(-9),mol/L外源性RA有效援救.RA缺乏时斑马鱼心脏表现出管状心脏、无向右环化或环化不完全、房室分化异常及房室管区血液反流.与野生型胚胎相比,DEAB处理后斑马鱼胚胎心率和心室收缩分数降低,心房利钠肽A基因表达改变,在心室部位表达清晰强烈,在心房部位表达明显减弱. 结论 DEAB影响胚胎发育有剂量依赖性和时效性,其致畸作用能被外源性RA有效拮抗.RA缺乏影响心脏早期发育的多个重要环节,导致心脏收缩功能受损.心脏心房利钠肽A基因表达受RA信号调控.
목적 이용신형모식생물반마어,채용외원성시황철탈경매2억제제--대이을안기분갑철(4-diethylaminobenzaldehyde,DEAB),건립유갑산(retinoic acid,RA)결핍적반마어모형,탐토기대반마어배태심장발육적영향. 방법 재반마어배태수정후5、8、10.3 h,분별용1×10~(-6),5×10~(-6)、10×10~(-6),25×10~(-6) mol/L적DEAB처리,재해부현미경하실시관찰배태발육적전과정,재수정후5 h급여1×10~(-9) mol/L외원성RA간예,관찰기대DEAB치기적길항작용.통과배태심장표형관찰、심솔화심실수축분수비교이급심장특이분자표기--심방리납태A기인정체원위잡교실험분석RA결핍대배태심장발육적영향. 결과 외원성DEAB처리후,배태생존솔수착처리농도증가이강저,수착처리시간점후이이승고.당DEAB농도≥5×10~(-6) mol/L시,반마어기태솔위100%,이상표형일치,병능피1×10~(-9),mol/L외원성RA유효원구.RA결핍시반마어심장표현출관상심장、무향우배화혹배화불완전、방실분화이상급방실관구혈액반류.여야생형배태상비,DEAB처리후반마어배태심솔화심실수축분수강저,심방리납태A기인표체개변,재심실부위표체청석강렬,재심방부위표체명현감약. 결론 DEAB영향배태발육유제량의뢰성화시효성,기치기작용능피외원성RA유효길항.RA결핍영향심장조기발육적다개중요배절,도치심장수축공능수손.심장심방리납태A기인표체수RA신호조공.
Objective To study the effect of retinal dehydrogenase type 2 inhibitor (4-diethylaminobenzaldehyde,DEAB) on embryonic CSrdiac develclpment of zebrafish model with retinoic acid(RA)deficiency. Methods Zebrafish embryos were treated with DEAB at various concentrations including 1×10~(-6),5×10~(-6),10×10~(-6),25×10~(-6)mol/L at 5,8 and 10.3 hours post fertilization,respectively.The effects of DEAB on the embryonic development were assessed under microscope.1×10~(-9)mol/L exogenous RA was then added to detect the antagonistic effect against DEAB.The abnormal cardiac phenotype,heart rate and ventricular systolic fraction were observed and analyzed between wild type and DEAB treated groups.The expression of specific cardiac gene, natriuretic peptide precursor A,was monitored by whole-mount in situ hybridization to demonstrate the effect of RA signaling on early cardiac development. Results The survival rate of zebrafish embryos declined with the increase of DEAB concentration at different developmental stage.The percentage of abnormal embryos reached 100% when DEAB over 5×10~(-6)mol/L.1×10~(-9) mol/L exogenous RA could eliminate the teratogenic effect of DEAB(≥5×10~(-6)mol/L).DEAB treated embryos presented abnormal cardiac phenotype,including tubular heart,incomplete D-loop,abnormal atrioventricular development,regurgitation,slow blood flow and weak heart beat.The difference of heart rate and ventrieular systolic fraction between wild type and RA deficiency embryos was of statistical significance(P<0.05).The natriuretic peptide precursor A expression remained in the ventricle,but reduced obviously in the atrium with RA signaling deficiency. Conclusions The effects of DEAB on the embryonic development are dose-dependent and time-dependent,and could be rescued by exogenous RA.RA signaling plays a critical role in several key stages of early cardiac development and natriuretie peptide precursor A expression.