CAJ | 학술논문

目的利用二维电泳分析乙醇诱导斑马鱼胚胎发育畸形的可能分子机制.方法采用400 mmol/L乙醇在斑马鱼胚胎圆顶期处理3 h,观察乙醇导致的斑马鱼胚胎发育畸形;通过二维凝胶电泳分析受精卵期、圆顶期、胚盾期和5-体节期的胚胎总蛋白,采用差减分析法减少母源性蛋白的干扰,比较5一体节期正常对照组和乙醇处理组斑马鱼胚胎蛋白表达谱的改变;通过原位杂交验证二维电泳的分析结果.结果 400 mmol/L乙醇处理导致62%的胚胎产生体轴发育障碍和60%的胚胎产生独眼畸形.二维电泳分析结果显示,400 mmol/L乙醇处理会导致受精后12 h(12 hours post fertilization,12 hpf)斑马鱼胚胎胶原蛋白2a1和TAR DNA结合蛋白表达分别下调81%和73%.随后原位杂交显示,胶原蛋白2a1在乙醇处理组胚胎体轴的表达明显下调;24 hpf乙醇处理胚胎胶原蛋白2a1在胚胎体轴的表达水平逐渐恢复正常,但神经管结构受到严重影响.结论乙醇处理后斑马鱼胚胎发育畸形可能是由于乙醇干扰了胚胎发育过程中胶原蛋白2a1和TAR DNA结合蛋白的表达;其中乙醇对胶原蛋白2a1在轴中胚层早期表达的干扰,可能是导致发育后期体轴和神经管畸形的重要原因.
목적 이용이유전영분석을순유도반마어배태발육기형적가능분자궤제.방법 채용400 mmol/L을순재반마어배태원정기처리3 h,관찰을순도치적반마어배태발육기형;통과이유응효전영분석수정란기、원정기、배순기화5-체절기적배태총단백,채용차감분석법감소모원성단백적간우,비교5일체절기정상대조조화을순처리조반마어배태단백표체보적개변;통과원위잡교험증이유전영적분석결과.결과 400 mmol/L을순처리도치62%적배태산생체축발육장애화60%적배태산생독안기형.이유전영분석결과현시,400 mmol/L을순처리회도치수정후12 h(12 hours post fertilization,12 hpf)반마어배태효원단백2a1화TAR DNA결합단백표체분별하조81%화73%.수후원위잡교현시,효원단백2a1재을순처리조배태체축적표체명현하조;24 hpf을순처리배태효원단백2a1재배태체축적표체수평축점회복정상,단신경관결구수도엄중영향.결론 을순처리후반마어배태발육기형가능시유우을순간우료배태발육과정중효원단백2a1화TAR DNA결합단백적표체;기중을순대효원단백2a1재축중배층조기표체적간우,가능시도치발육후기체축화신경관기형적중요원인.
Objective To study the putative mechanisms underlying fetal alcohol syndrome by comparative protein-profile analysis between normal and ethanol-treated zebrafish embryo with twodimensional electrophoresis (2-DE).Methods Zebrafish embryos were exposed in 400 mmol/L ethanol at dome stage for 3 hours,and then ethanol-induced abnormalities were observed.Proteomes of zebrafish embryos at early stages including zygote stage,dome stage,shield stage and 5-somite stage,were separated by 2-DE.The subtraction analysis method was applied to eliminate the interference from maternal derived proteins.The ethanol-treated embryos at 5-somite stage was analyzed by 2-DE,and the protein profile was compared with that generated from control embryos at the same stage.The data obtained from 2-DE analysis were verified by in-situ hybridization.Results 400 mmol/L ethanol treatment caused axial malformation (62%) and cyclopia (60%) in zebrafish embryos.The 2-DE analysis showed that the expression of Collagen2al (Col2a1) and TAR DNA binding protein (TDP) was decreased in 12 hours post fertilization (12 hpf) ethanol-treated embryos by 81% and 73%,respectively.The in-situ hybridization also demonstrated that the expression of Col2al in axial mesoderm was reduced by ethanol treatment at the same stage.But for 24 hpf ethanoltreated embryos,the expression of Col2al in axis recovered to a comparable level to that in control embryos,while the structure of neural tube was disrupted severely by ethanol exposure.Conclusions It is suggested that the expressions of Col2al and TDP were disrupted by ethanol during early stage,which might induce the zebrafish developmental abnormalities.The ethanol interference on early expression of Col2al is supposed to be one of the major reasons leading to later abnormalities of axis and neutral tube.