叶酸缺乏对斑马鱼胚胎造血系统影响的初步研究  

EFFECT OF FOLIC ACID DEFICIENCY ON ZEBRAFISH HEMATOPOIESIS

作　　者：孙淑娜[1] 桂永浩[1] 蒋璆[2] 宋后燕[2] 钟涛 

机构地区：[1]复旦大学儿科医院,上海201102 [2]复旦大学分子医学教育部重点实验室,上海200032 [3]美国Vanderbilt大学医学院细胞生物学实验室

出　　处：《营养学报》2010年第1期21-25,共5页Acta Nutrimenta Sinica

基　　金：国家教育部博士点基金(No.200802461111)

摘　　要：目的观察叶酸缺乏对斑马鱼胚胎造血干细胞发育的干扰作用以及对胚胎造血区凋亡的影响,初步探讨叶酸缺乏导致血液生成异常的机制。方法将二氢叶酸还原酶基因(DHFR)功能阻断构建叶酸缺乏斑马鱼模型,分别用二氢叶酸还原酶抑制剂甲氨蝶呤(MTX)以及DHFR表达阻抑技术处理斑马鱼胚胎,造成胚胎体内叶酸缺乏的模式。在显微镜下观察胚胎发育情况,用胚胎固兰染色观察斑马鱼胚胎血细胞生成量,并用原位杂交和Real-timePCR方法检测血细胞分化和生成密切相关基因FLK-1、GATA1和GATA2的表达情况,利用TUNEL法检测斑马鱼胚胎造血区的凋亡状况。结果与各对照组相比,叶酸缺乏组斑马鱼胚胎的造血区形态学发育异常,血细胞数量明显减少,FLK-1、GATA1和GATA2的表达显著下降,胚胎造血区的凋亡增加。结论叶酸缺乏干扰了血细胞的生成,叶酸缺乏组胚胎FLK-1、GATA1和GATA2的表达降低和造血区的凋亡增加与胚胎血液系统发育异常相关。Objective To observe the development of hematopoietic stem cell and the apoptosis of ICM （intermediate cell mass） in folic acid deficient zebrafish embryos and investigate the mechanism by which folic acid deficiency induces abnormal hematopoiesis. Method The folic acid deficient zebrafish model was induced by both using the dihydrofolate reductase antagonism methotrexate （MTX） and knock-down dihydrofolate reductase gene. The development of embryos was observed under microscope. The blood cells were detected by O-dianisidine staining. Whole-mount in situ hybridization and real-time PCR were performed to examine the expression of FLK-1, GATAland GATA2. Apoptosis in intermediate cell mass （ICM） was examined by TUNEL （terminal -deoxynucleotidyl transferase mediated nick end labeling） method. Results The abnormal developments of ICMs were observed both in MTX treated embryos and DHFR knock-down embryos. O-dianisidine staining revealed that folic acid deficiency resulted in the decreasing number of blood cells. In folic acid deficient embryos, the expression of FLK-1, GATAland GATA2 was reduced and the apoptosis in ICMs was increased. Conclusion The abnormal hematopoiesis in zebrafish induced by folic acid deficiency is related with the increasing apoptosis in ICMs and decreasing expressions of FLK-1, GATAland GATA2.

关 键 词：叶酸 斑马鱼 血液系统 

分 类 号：Q4[生物学—生理学]