二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用  

Effects of Dihydrofolate Reductase Gene on the Development of Nervous System in 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,上海201102 [2]复旦大学分子医学教育部重点实验室,上海200032

出　　处：《中国实验动物学报》2010年第2期127-130,I0006,I0007,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家教育部博士点基金资助(编号:200802461111)

摘　　要：目的观察二氢叶酸还原酶基因(DHFR)功能阻抑斑马鱼胚胎的颅脑部发育情况,初步探讨二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用。方法采用显微注射吗啡啉修饰的反义核苷酸(MO)的方法进行DHFR表达阻抑。胚胎发育至受精后48hpf观察胚胎的颅部发育情况,在60hpf时经石蜡切片进一步观察胚胎的脑发育状况。利用胚胎整体原位杂交的方法检测影响神经系统发育的关键因子ngn1和huc的表达情况。结果显微注射MO可成功的进行DHFR表达阻抑。DHFR表达阻抑组胚胎存在颅脑部发育明显异常和ngn1、huc的表达强度明显减弱,且与显微注射的MO剂量呈正相关。结论DHFR在斑马鱼颅脑发育中有重要作用;其功能阻抑可导致胚胎颅脑部发育异常,其机理与ngn1和huc的的表达减弱有关。Objective To observe the development of cranium and brain in dihydrofolate reductase gene-inhibited zebrafish embryos and to investigate the mechanism of action of dihydrofolate reductase gene（DHFR） in this process.Methods DHFR was inhibited by microinjecting morpholino oligonucleotides（MO）.The development of heads at 48 hours post fertilization（48 hpf） was observed and the development of the brain at 60 hpf was examined by histopathology.Whole-mount in situ hybridization was performed to detect the expression of ngn1 and huc,which are two key factors in the developments of nervous system.Results DHFR was successfully inhibited by microinjecting MO.The abnormal developments of heads and brains were observed in DHFR-inhibited embryos.Compared with controls,the expressions of ngn1 and huc were reduced in DHFR-inhibited groups.With the increasing doses of MOs,the malformations were more severe and the reduction of ngn1 and huc expressions was more pronounced.Conclusion DHFR plays a major role in the development of nervous system in zebrafish.The malformation of cranium and brain induced by DHFR-inhibition was related with the decreasing expression of ngn1 and huc.

关 键 词：二氢叶酸还原酶基因 神经系统 斑马鱼 

分 类 号：Q3[生物学—遗传学] Q7