叶酸缺乏对斑马鱼体轴发育的影响  

The effects of folic acid deficiency on the axial development of zebrafish

作　　者：孙淑娜[1] 桂永浩[1] 蒋璆[2] 钱林溪[2] 宋后燕[2] 

机构地区：[1]复旦大学附属儿科医院心血管中心,上海201102 [2]复旦大学上海医学院生物化学与分子生物学系-分子医学教育部重点实验室,上海200032

出　　处：《复旦学报（医学版）》2009年第6期663-669,共7页Fudan University Journal of Medical Sciences

基　　金：国家教育部博士点基金(200802461111)项目;2007年复旦大学上海医学院"基础-临床交叉研究基金"项目

摘　　要：目的构建叶酸缺乏斑马鱼模型,观察叶酸缺乏斑马鱼胚胎的体轴发育情况,初步探讨叶酸缺乏导致胚胎体轴发育异常的机制。方法分别应用二氢叶酸还原酶(dhfr)抑制剂甲氨蝶呤(MTX)以及二氢叶酸还原酶(dhfr)基因阻抑技术,通过下调二氢叶酸还原酶功能构建叶酸缺乏斑马鱼模型。在胚胎发育至17 hpf时在显微镜下观察胚胎的体轴偏移情况。利用特异性探针通过胚胎整体原位杂交的方法标记肝脏、胰脏以及心脏,以观察其位于中线两侧的位置。利用胚胎整体原位杂交和Real-time PCR的方法检测与体轴发育密切相关的Nodal信号通路下游因子ntl和gsc的表达情况。结果叶酸缺乏组一定比例胚胎存在体轴偏移以及肝脏、胰脏和心脏左右轴异位。叶酸缺乏组Nodal信号通路下游因子ntl以及gsc的表达强度无明显变化,但表达的空间模式发生改变,提示轴中胚层发育紊乱。结论叶酸缺乏可导致斑马鱼胚胎体轴发育异常以及轴中胚层发育紊乱,但对Nodal信号通路无明显干扰作用。Objective To construct a folic acid deficient model in zebrafish and to observe the axial development in folie acid deficient embryos, so as to probe the mechanism by which folic acid deficiency induces abnormal development of axis. Methods We constructed the folic acid deficient zebrafish model by both using the antagonism of dihydrofolate reductase （MTX） and knocking-down dihydrofolate reductase gene. Then we observed the axial excursion of folic acid deficient embryos at 17 hpf under microscope. We labeled and observed the positions of liver, spleen and heart by using whole-mount in situ hybridization with specific antisense RNA probes. The expressions of some genes, which are down stream factors of Nodal signal pathway and important for axial development, were detected by whole-mount in situ hybridization and Real-time PCR. Results Parts of folic acid deficient embryos had axial excursion and abnormal positions of liver, spleen and heart. The expressing intensities of ntl and gsc appeared normal in folic acid deficient embryos, but the expressing spatial patterns were abnormal, which revealed the malformation of axial mesoderm. Conclusions Folic acid deficiency induced the abnormal development of axis and the malformation of axial mesoderm. Folic acid deficiency had no obviouse effect on Nodal pathway.

关 键 词：叶酸 二氢叶酸还原酶 体轴 Nodal信号通路 

分 类 号：Q132.7[生物学—普通生物学]