二维电泳分析胎儿酒精综合征斑马鱼模型蛋白表达谱  

Two-dimensional electrophoresis analysis for protein profile change in zebrafish alcohol syndrome model

作　　者：钱林溪[1] 孙淑娜[2] 蔡威[1] 王跃祥[3] 蒋缪[3] 宋后燕[3] 

机构地区：[1]上海交通大学医学院附属新华医院上海市儿科医学研究所,200092 [2]复旦大学附属儿科医院 [3]复旦大学分子医学教育部重点实验室

出　　处：《中华围产医学杂志》2011年第5期283-288,共6页Chinese Journal of Perinatal Medicine

基　　金：国家自然科学基金杰出青年科学基金（30328009）;复旦大学“十五”“211工程”重点学科建设项目;上海交通大学医学院青年基金（YZ1011）

摘　　要：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400mmol／L乙醇在斑马鱼胚胎圆顶期处理3h，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-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400mmol／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，400mmol／L乙醇处理会导致受精后12h（12hourspostfertilization，12hpf）斑马鱼胚胎胶原蛋白2al和TARDNA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2al在乙醇处理组胚胎体轴的表达明显下调；24hpf乙醇处理胚胎胶原蛋白2al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2al和TARDNA结合蛋白的表达；其中乙醇对胶原蛋白2al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。Objective To study the putative mechanisms underlying fetal alcohol syndrome by comparative protein profile analysis between normal and ethanol treated zebrafish embryo with two dimensional 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 zehrafish 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 （Col2al） 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.

关 键 词：斑马鱼 胎儿酒精综合征 蛋白质组 电泳 凝胶 双向 

分 类 号：R-332[医药卫生]