叶酸缺乏对斑马鱼背主动脉发育的影响

Effects of Folic Acid-Deficiency on the Development of Dorsal Artery in Zebrafish

目的观察叶酸缺乏斑马鱼胚胎的背主动脉(DA)发育情况,初步探讨叶酸缺乏后胚胎DA发育异常的机理。方法采用将二氢叶酸还原酶(DHFR)功能阻断的方法构建叶酸缺乏斑马鱼模型,分别应用DHFR抑制剂甲氨蝶呤(MTX)以及DHFR基因knock-down技术处理斑马鱼胚胎。在胚胎发育至48 hpf时在显微镜下观察胚胎的整体发育情况,在60 hpf时应用荧光显微造影的方法观察胚胎的背主动脉发育状况。利用胚胎整体原位杂交和real-time PCR的方法检测影响DA发育的关键因子ephrinB2、Ang-1和Radar的表达情况,利用TUNEL法检测胚胎底索的凋亡情况。结果MTX处理组胚胎以及DHFR knock-down组胚胎有相似的胚胎发育异常表型。荧光显微造影显示叶酸缺乏组胚胎的DA发育异常。叶酸缺乏组胚胎的ephrinB2、Ang-1和Radar表达减弱,底索凋亡增加。结论叶酸缺乏可导致斑马鱼胚胎背主动脉发育异常,其机理与ephrinB2、Ang-1和Radar的表达减弱以及底索凋亡增加有关。

Objective Tn observe the development of dorsal artery （DA） in folic acid-deficient zebrafish embryos and investigate the mechanism by which folic acid deficiency induces DA malformation. Methods The folic acid-deficient zebrafish model was established by using both dihydrofolate reductase antagonism MTX and dihydrofolate reductase gene knock-down. The over-all development of embryos was observed under the microscope at 48 hpf and the development of DA was assessed by fluorescein microangiography at 60 hpf. Whole-mount in situ hybridization and real-time PCR were performed to detect the expression of ephrinB2, Ang-1 and Radar. Apoptosis in the hypochord was examined by TUNEL （terminal-deoxynucleotidyl transferase mediated nick end labeling） staining. Results Similarly abnormal phenotypes were observed both in MTX treated embryos and DHFR knock-down embryos. Fluorescein microangiography revealed that folic acid deficiency affected the development of DA. In folic acid deficient embryos,the expression of ephrinB2, Ang-1 and Radar were reduced and apoptosis was increased. Conclusion The malformation of DA in zebrafish induced by folic acid deficiency is associated with the increasing apoptosis in hypochord and decreasing expression of ephriaB2,Ang-1 and Radar.