胰島素樣生長因子結(jié)合蛋白-2參與斑馬魚胚胎心血管系統(tǒng)發(fā)育的實驗研究
發(fā)布時間：2019-05-20 07:38
【摘要】：目的通過顯微注射嗎啡啉修飾的反義寡核苷酸制作斑馬魚IGFBP-2基因表達下調(diào)的動物模型,觀察IGFBP-2表達下調(diào)導(dǎo)致斑馬魚胚胎心臟血管發(fā)育的異常表型及其對心臟發(fā)育相關(guān)基因表達的影響。方法胚胎整體原位雜交驗證IGFBP-2基因在斑馬魚胚胎發(fā)育早期的時空表達譜。特異抑制IGFBP-2基因啟動子的嗎啡啉(morpholino,MO)和標準對照嗎啡啉(Con-MO)由Gene-Tools公司設(shè)計合成。設(shè)置0.05、0.10、0.25和1.0 mmol/L 4個不同濃度梯度的IGFBP-2 MO注射組,觀察不同注射濃度對斑馬魚胚胎心臟異常表型發(fā)生率和死亡率的影響,并與Con-MO注射組以及野生型(Wild type,Wt)對照組比較。詳細記錄0.25 mmol/L濃度IGFBP-2 MO注射組不同發(fā)育時段斑馬魚胚胎心臟發(fā)育的異常表型。熒光顯微鏡下觀察IGFBP-2 EGFP重組質(zhì)粒單獨以及與IGFBP-2 MO或Con-MO共注射后12hpf胚胎的綠色熒光表達。原位雜交比較IGFBP-2 MO注射組與Wt組斑馬魚心房標記基因Amhc的表達情況;觀察IGFBP-2基因下調(diào)的Vmhc-EGFP轉(zhuǎn)基因斑馬魚心室特異性綠色熒光的變化;顯微血管熒光造影顯示IGFBP-2 MO注射組與Wt組胚胎外周血管發(fā)育的差異。結(jié)果斑馬魚胚胎早期的發(fā)育過程中,IGFBP-2基因在眼球、中腦和肝臟先后表達。0.25 mmol/L濃度MO注射組12hpf存活的胚胎在48hpf有59.6%發(fā)生心臟發(fā)育的異常表型,包括心管搏動無力、心包水腫和房室環(huán)化障礙,部分胚胎發(fā)生心房擴大、房室反流伴循環(huán)瘀滯,且畸形隨發(fā)育時間推移加重。接受單獨顯微注射IGFBP-2EGFP重組質(zhì)粒和與Con-MO共同注射的Wt胚胎12hpf出現(xiàn)明顯的綠色熒光表達,而重組質(zhì)粒與IGFBP-2MO共同注射的胚胎幾乎無熒光表達,證實MO下調(diào)斑馬魚胚胎IGFBP-2基因的有效性。48hpf胚胎整體原位雜交顯示IGFBP-2 MO注射組心房特異標記基因Amhc的表達下調(diào);48hpf IGFBP-2基因下調(diào)的Vmhc-EGFP轉(zhuǎn)基因斑馬魚心室特異綠色熒光蛋白的表達減弱;60hpf IGFBP-2 MO注射組顯微血管熒光造影顯示體節(jié)間血管顯影稀疏紊亂。結(jié)論顯微注射IGFBP-2 MO能夠有效實現(xiàn)斑馬魚胚胎的IGFBP-2基因下調(diào)。IGFBP-2基因下調(diào)導(dǎo)致斑馬魚胚胎心臟血管的異常表型,并抑制心臟發(fā)生基因Amhc和Vmhc的表達。IGFBP-2在斑馬魚胚胎心血管系統(tǒng)的正常發(fā)育過程中起到重要作用。
[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.
【作者單位】： 復(fù)旦大學(xué)附屬兒科醫(yī)院心血管中心;復(fù)旦大學(xué)分子醫(yī)學(xué)教育部重點實驗室;
【分類號】：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.
【作者單位】： 復(fù)旦大學(xué)附屬兒科醫(yī)院心血管中心;復(fù)旦大學(xué)分子醫(yī)學(xué)教育部重點實驗室;
【分類號】：Q344
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