胰岛素样生长因子结合蛋白-2参与斑马鱼胚胎心血管系统发育的实验研究  

The experimental research for contribution of insulin-like growth factor binding protein-2 during zebrafish embryonic cardiovascular development

作　　者：高珊[1] 王薇[1] 梁进涛[1] 桂永浩[1] 蒋璆[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]复旦大学分子医学教育部重点实验室,上海200032

出　　处：《复旦学报（医学版）》2010年第1期43-51,共9页Fudan University Journal of Medical Sciences

摘　　要：目的通过显微注射吗啡啉修饰的反义寡核苷酸制作斑马鱼IGFBP-2基因表达下调的动物模型,观察IGFBP-2表达下调导致斑马鱼胚胎心脏血管发育的异常表型及其对心脏发育相关基因表达的影响。方法胚胎整体原位杂交验证IGFBP-2基因在斑马鱼胚胎发育早期的时空表达谱。特异抑制IGFBP-2基因启动子的吗啡啉(morpholino,MO)和标准对照吗啡啉(Con-MO)由Gene-Tools公司设计合成。设置0.05、0.10、0.25和1.0 mmol/L 4个不同浓度梯度的IGFBP-2 MO注射组,观察不同注射浓度对斑马鱼胚胎心脏异常表型发生率和死亡率的影响,并与Con-MO注射组以及野生型(Wild type,Wt)对照组比较。详细记录0.25 mmol/L浓度IGFBP-2 MO注射组不同发育时段斑马鱼胚胎心脏发育的异常表型。荧光显微镜下观察IGFBP-2 EGFP重组质粒单独以及与IGFBP-2 MO或Con-MO共注射后12hpf胚胎的绿色荧光表达。原位杂交比较IGFBP-2 MO注射组与Wt组斑马鱼心房标记基因Amhc的表达情况;观察IGFBP-2基因下调的Vmhc-EGFP转基因斑马鱼心室特异性绿色荧光的变化;显微血管荧光造影显示IGFBP-2 MO注射组与Wt组胚胎外周血管发育的差异。结果斑马鱼胚胎早期的发育过程中,IGFBP-2基因在眼球、中脑和肝脏先后表达。0.25 mmol/L浓度MO注射组12hpf存活的胚胎在48hpf有59.6%发生心脏发育的异常表型,包括心管搏动无力、心包水肿和房室环化障碍,部分胚胎发生心房扩大、房室反流伴循环瘀滞,且畸形随发育时间推移加重。接受单独显微注射IGFBP-2EGFP重组质粒和与Con-MO共同注射的Wt胚胎12hpf出现明显的绿色荧光表达,而重组质粒与IGFBP-2MO共同注射的胚胎几乎无荧光表达,证实MO下调斑马鱼胚胎IGFBP-2基因的有效性。48hpf胚胎整体原位杂交显示IGFBP-2 MO注射组心房特异标记基因Amhc的表达下调;48hpf IGFBP-2基因下调的Vmhc-EGFP转基因斑马鱼心室特异绿色荧光蛋白的表达减弱;60hpf IGFObjective To establish a zebrafish IGFBP-2 gene knock-down model by morphilino modified antisense oligonucleotide injection,so as to investigate the abnormal phenotypes of heart and vessels in early stage of zebrafish development and the expression of zebrafish cardiogenesis related genes. Methods The spatiotemporal expression of IGFBP-2 gene in early stage of zebrafish development was testified by whole mount in situ hybridization with antisense RNA IGFBP-2 probe.The IGFBP-2 morpholino（IGFBP-2 MO） that especially inhibited the gene promoter and standard control morpholino（Con-MO） were designed and synthesized by Gene-tools Corporation.Four different concentration gradients（0.05,0.10,0.25 and 1.0 mmol/L） were set as IGFBP-MO injection groups with 0.25 mmol/L Con-MO injection group and wild type group as controls.Contribution to the incidence of heart abnormal phenotypes and mortality rate induced by 4 different IGFBP-2 concentrations injection group was recorded and compared with 2 control groups.Heart abnormal phenotypes at different developmental stages in 0.25 mmol/L IGFBP-2 injection group were observed in detail.To validate the effectiveness of IGFBP-2 MO,the expression of enhanced green fluorescence presented by wild type zebrafish embryos at 12hpf which received single injection of IGFBP-2 EGFP recombinant plasmid and those co-injected with Con-MO or IGFBP-2 MO were detected.To investigate the regulation relationship between IGFBP-2 gene and other cardiogenesis related genes,expression of atrium specific marker gene Amhc was detected in IGFBP-2 MO and wild type group by in situ hybridization.Ventricle specific green fluorescence of Vmhc-EGFP transgenic zebrafish embryos whose IGFBP-2 gene was knocked-down were compared with those untreated.Zebrafish peripheral vascular development in the IGFBP-2 MO group was also checked out by micro-angiography. Results Whole mount in situ hybridization revealed that IGFBP-2 gene expressed in turn at eyes,midbrain and then focused on liver in early stage of ze

关 键 词：IGFBP-2 斑马鱼 胚胎 心血管发育 

分 类 号：Q132.4[生物学—普通生物学]