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
发布时间：2019-05-20 07:38
【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 IGFBP-2 MO注射组显微血管荧光造影显示体节间血管显影稀疏紊乱。结论显微注射IGFBP-2 MO能够有效实现斑马鱼胚胎的IGFBP-2基因下调。IGFBP-2基因下调导致斑马鱼胚胎心脏血管的异常表型,并抑制心脏发生基因Amhc和Vmhc的表达。IGFBP-2在斑马鱼胚胎心血管系统的正常发育过程中起到重要作用。
[Abstract]:Objective to establish an animal model of down-regulation of IGFBP-2 gene expression in zebrafish by microinjection of morphine modified antisense oligodeoxynucleotides. To observe the abnormal phenotype of cardiac vascular development in zebrafish embryos induced by the down-regulation of IGFBP-2 expression and its effect on the expression of cardiac development related genes. Methods the temporal and spatial expression profile of IGFBP-2 gene in the early embryonic development of zebrafish was verified by in situ hybridization. Morphine (morpholino,MO) and standard control morphine (Con-MO), which specifically inhibit the promoter of IGFBP-2 gene, were designed and synthesized by Gene-Tools Company. Four IGFBP-2 MO injection groups with different concentration gradients of 0.05, 0.10, 0.25 and 1.0 mmol/L were set up to observe the effects of different injection concentrations on the incidence and mortality of abnormal heart phenotype in zebrafish embryos. It was compared with Con-MO injection group and wild type (Wild type,Wt) control group. The abnormal phenotypes of embryonic heart development of zebrafish at different developmental stages in 0.25 mmol/L IGFBP-2 MO injection group were recorded in detail. The green fluorescence expression of 12hpf embryos after IGFBP-2 EGFP recombinant plasmid and co-injection with IGFBP-2 MO or Con-MO was observed under fluorescence microscope. The expression of atrial marker gene Amhc in zebrafish in IGFBP-2 MO injection group and Wt group was compared by in situ hybridization, and the changes of ventricular specific green fluorescence in Vmhc-EGFP transgenic zebrafish with down-regulated IGFBP-2 gene were observed. Microfluorescein angiography showed the difference of embryonic peripheral vascular development between IGFBP-2 MO injection group and Wt group. Results during the early development of zebrafish embryos, IGFBP-2 gene was expressed successively in eyeball, midbrain and liver. 59.6% of 12hpf survival embryos in MO injection group had abnormal phenotype of cardiac development in 48hpf. These include ventricular pulsatile weakness, pericardial edema and atrioventricular cyclization disorder, atrial enlargement in some embryos, atrioventricular reflux with circulatory stasis, and malformation aggravated with the passage of development time. The 12hpf of Wt embryos injected with IGFBP-2EGFP alone and Wt injected with Con-MO showed obvious green fluorescence expression, while there was almost no fluorescence expression in the embryos injected with IGFBP-2MO. It was confirmed that MO could down-regulate the IGFBP-2 gene of zebrafish embryos. The expression of atrial specific marker gene Amhc was down-regulated in 48hpf embryos. The expression of ventricular specific green fluorescent protein in Vmhc-EGFP transgenic zebrafish with down-regulated 48hpf IGFBP-2 gene was decreased, and microfluorescein in 60hpf IGFBP-2 MO injection group showed sparse Internode vascular development disorder. Conclusion Microinjection of IGFBP-2 MO can effectively down-regulate the IGFBP-2 gene of zebrafish embryos. The down-regulation of IGFBP-2 gene leads to the abnormal phenotype of cardiac blood vessels in zebrafish embryos. IGFBP-2 plays an important role in the normal development of cardiovascular system in zebrafish embryos.
【作者单位】： 复旦大学附属儿科医院心血管中心;复旦大学分子医学教育部重点实验室;
【分类号】：Q344
[Abstract]:Objective to establish an animal model of down-regulation of IGFBP-2 gene expression in zebrafish by microinjection of morphine modified antisense oligodeoxynucleotides. To observe the abnormal phenotype of cardiac vascular development in zebrafish embryos induced by the down-regulation of IGFBP-2 expression and its effect on the expression of cardiac development related genes. Methods the temporal and spatial expression profile of IGFBP-2 gene in the early embryonic development of zebrafish was verified by in situ hybridization. Morphine (morpholino,MO) and standard control morphine (Con-MO), which specifically inhibit the promoter of IGFBP-2 gene, were designed and synthesized by Gene-Tools Company. Four IGFBP-2 MO injection groups with different concentration gradients of 0.05, 0.10, 0.25 and 1.0 mmol/L were set up to observe the effects of different injection concentrations on the incidence and mortality of abnormal heart phenotype in zebrafish embryos. It was compared with Con-MO injection group and wild type (Wild type,Wt) control group. The abnormal phenotypes of embryonic heart development of zebrafish at different developmental stages in 0.25 mmol/L IGFBP-2 MO injection group were recorded in detail. The green fluorescence expression of 12hpf embryos after IGFBP-2 EGFP recombinant plasmid and co-injection with IGFBP-2 MO or Con-MO was observed under fluorescence microscope. The expression of atrial marker gene Amhc in zebrafish in IGFBP-2 MO injection group and Wt group was compared by in situ hybridization, and the changes of ventricular specific green fluorescence in Vmhc-EGFP transgenic zebrafish with down-regulated IGFBP-2 gene were observed. Microfluorescein angiography showed the difference of embryonic peripheral vascular development between IGFBP-2 MO injection group and Wt group. Results during the early development of zebrafish embryos, IGFBP-2 gene was expressed successively in eyeball, midbrain and liver. 59.6% of 12hpf survival embryos in MO injection group had abnormal phenotype of cardiac development in 48hpf. These include ventricular pulsatile weakness, pericardial edema and atrioventricular cyclization disorder, atrial enlargement in some embryos, atrioventricular reflux with circulatory stasis, and malformation aggravated with the passage of development time. The 12hpf of Wt embryos injected with IGFBP-2EGFP alone and Wt injected with Con-MO showed obvious green fluorescence expression, while there was almost no fluorescence expression in the embryos injected with IGFBP-2MO. It was confirmed that MO could down-regulate the IGFBP-2 gene of zebrafish embryos. The expression of atrial specific marker gene Amhc was down-regulated in 48hpf embryos. The expression of ventricular specific green fluorescent protein in Vmhc-EGFP transgenic zebrafish with down-regulated 48hpf IGFBP-2 gene was decreased, and microfluorescein in 60hpf IGFBP-2 MO injection group showed sparse Internode vascular development disorder. Conclusion Microinjection of IGFBP-2 MO can effectively down-regulate the IGFBP-2 gene of zebrafish embryos. The down-regulation of IGFBP-2 gene leads to the abnormal phenotype of cardiac blood vessels in zebrafish embryos. IGFBP-2 plays an important role in the normal development of cardiovascular system in zebrafish embryos.
【作者单位】： 复旦大学附属儿科医院心血管中心;复旦大学分子医学教育部重点实验室;
【分类号】：Q344
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