【作者】 高珊；

【导师】 桂永浩； 宋后燕； 杨毅； 于敏；

【作者基本信息】 复旦大学 ， 心血管内科， 2009， 博士

【摘要】 胰岛素样生长因子结合蛋白(Insulin-like growth factor binding proteins, IGFBPs)是一组在生物进化过程中高度保守的分泌蛋白家族,属于胰岛素样生长因子信号传导系统超家族成员。IGFBPs通过调节循环中胰岛素样生长因子的半衰期及其生物活性发挥其内分泌调控作用。IGFBPs在胚胎期多种器官组织中高度表达,提示其可能在胚胎发育的过程中扮演重要角色。已有研究证实IGFBPs在体外可以诱导促进特定系细胞株向成熟的心肌和血管内皮细胞分化,IGFBP-4对于爪蟾胚胎的心脏发生具有正向调节作用。进一步探索IGFBPs在胚胎心血管系统发育中的作用将有助于我们更好的理解人类出生缺陷特别是先天性心脏病的发病机理和分子机制,为更有效地预防控制疾病提供新的思路。近年来斑马鱼已成为研究脊椎动物胚胎早期发育,特别是心血管系统和心脏发生的理想模式动物。首先,斑马鱼的基因表达图谱已经明确,心血管系统发育过程与哺乳动物比较存在高度的相似性；其次,斑马鱼体外受精发育、早期通体透明,借助光学显微镜可以清楚实时地观察到心脏的发育情况；最重要的是,由于可以通过渗透作用从外界环境中被动获取氧,因此即使其循环系统存在严重畸形的情况下也能够存活较长时间。上述原因使斑马鱼在胚胎心血管系统发育的研究中具备独特的优势。第一部分胰岛素样生长因子结合蛋白-2在进化中高度保守以及IGFBP-2基因在斑马鱼胚胎发育早期的时空表达谱利用生物信息学方法证明生物进化过程IGFBP-2基因及蛋白的结构和功能高度保守。利用反义RNA探针标记的胚胎整体原位杂交技术分析IGFBP-2基因在斑马鱼胚胎发育早期的时空表达谱。IGFBP-2基因的杂交信号在野生型斑马鱼胚胎的双侧眼球、中枢神经系统(主要在中脑)和原始肝脏先后出现,72hpf以后主要集中表达于胚胎肝脏,而发育早期在胚胎心血管局部并无表达。第二部分建立IGFBP-2 MO基因表达下调的斑马鱼模型以及IGFBP-2下调后斑马鱼胚胎心脏发育的异常表型设计合成吗啡啉修饰的反义寡核苷酸特异性抑制IGFBP-2基因的翻译起始位点,通过MO显微注射技术建立斑马鱼胚胎IGFBP-2基因表达下调的模型。详细观察记录48和72hpf野生型组、Con-MO组以及IGFBP-2 MO不同注射剂量组之间斑马鱼胚胎的发育情况和出现心脏异常表型的比例。0.25mmol／L MO注射组胚胎自24hpf起依次出现心率下降、心包水肿、心房扩张、心室搏动无力,房室环化障碍等心脏畸形,部分胚胎存在不同程度的循环瘀滞和瓣膜反流。IGFBP-2 MO的注射剂量与胚胎心脏的异常表型存在剂量—效应关系。接受IGFBP-2 MO与IGFBP-2 EGFP重组质粒共注射的胚胎12hpf绿色荧光蛋白表达明显减弱,而野生型和Con-MO组的荧光蛋白表达正常。该结果验证了IGFBP-2MO的有效性,即其能够针对抑制斑马鱼胚胎内源性IGFBP-2的基因表达。第三部分IGFBP-2 MO基因下调对斑马鱼胚胎心脏发育标志基因表达、外周血管发育以及对Vmhc-EGFP转基因鱼心室特异绿色荧光蛋白表达的影响既然IGFBP-2表达下调严重干扰斑马鱼胚胎的正常心脏发育,我们将进一步研究其对斑马鱼胚胎心脏发育标志基因的表达和外周血管发育的影响作用。胚胎原位杂交结果显示心房特异性标志基因Amhc的表达下调；IGFBP-2基因下调的Vmhc-EGFP转基因斑马鱼48hpf心室特异性绿色荧光表达减弱；显微荧光血管造影显示IGFBP-2 MO组胚胎60hpf体节间血管显影稀疏紊乱,提示IGFBP-2同样参与斑马鱼胚胎外周血管的正常发育。更多还原

【Abstract】 The Insulin-like Growth Factor Binding Proteins(IGFBPs) constitute a secretary protein family of evolutionarily highly-conserved proteins which serve an endocrine function by regulating half-life time of circulating IGFs and modulating cellular responses to IGF signaling. IGFBPs genes abundantly expressed in many tissues and organs during embryonic developmental stage, which may suggested to play a critical role in the process of embryogenesis. It has been validated that IGFBPs are required for the induction of special cells strain into mature cardiomyocytes or vascular endothelial cells in vitro and IGFBP-4 is also a positive regulator to embryonic cardiogenesis of Xenopus in vivo. Further investigation to more precise function of IGFBPs in the process of embryonic cardiovascular system development can surely get a better understanding to the mystery of human born defect, particularly to the pathogenesis and molecule mechanisms of Congenital heart disease, thus lead us to some brand new ideas and more effective ways to prevent and manage this life-threatening disease.In recent years, zebrafish has emerged as an ideal animal model for studying vertebrate development and it is especially suitable for early stage development of cardiovascular system. First, gene expression map of zebrafish has already been identified and the development process of its cardiovascular system shared great similarity with mammals and human. Second, embryos of this species fertilize and develop externally, this optical clarity permits easy and real-time visualization of heart development; More importantly, zebrafish embryos are almost independent on well-developed and fully-functional cardiovascular system at early developmental stage. Without blood circulation even heart beat, zebrafish embryos can still take enough oxygen by passive diffusion to survive and continue to develop in a relatively normal way in the first week time. These unique advantages make zebrafish an outstanding model animal for the investigation to embryonic cardiovascular development and cardiogenesis. 复旦大学博士学位论文英文摘要PartⅠThe Evolutional Conservatism of Insulin-like Growth Factor Binding Protein-2 & The Spatiotemporal Expression of IGFBP-2 Gene during Early Stage of Zebrafish Embryonic DevelopmentTo prove the structure and function of IGFBP-2 gene and protein are extremely conserved during biological evolutionary process by bioinformatics method. Whole mount in situ hybridization with antisense RNA probe revealed the spatiotemporal expression pattern of IGFBP-2 gene in early stage of zebrafish embryonic development. IGFBP-2 gene expressed in turn at eyes, central nervous system (mainly at midbrain) and primitive liver. After 72hpf, IGFBP-2 gene predominantly expressed at zebrafish embryos’livers. There was no IGFBP-2 gene expression at local developing cardiovascular system.PartⅡEstablish IGFBP-2 Gene Morpholino Knock-down Zebrafish Model & The Heart Abnormal Phenotype Induced by IGFBP-2 Gene Down-regulationTo investigate the impact of IGFBP-2 gene down-regulation on the development of zebrafish embryonic heart and vasculature, well designed and synthesized morpholino modified antisense oligonucleotide which specific inhibits the initiating site of IGFBP-2 gene translation was microinjected into zebrafish embryos at one to four cells stage to block the IGFBP-2 gene expression. Four different concentration gradients:0.05,0.10,0.25 and 1.0mmol/L were set as IGFBP-2 MO injection groups with 0.25mmol/L Standard Control Morpholino (Con-MO) injection group and Wild type(Wt) group as controls.Contribution to the incidence of heart abnormal phenotypes and mortality rate induced by 4 different IGFBP-2 concentration injection group was recorded and compared with 2 control groups. The hearts of zebrafish embryos in IGFBP-2 MO group had defects in cardiac morphology and contractility in an IGFBP-2 Morpholino dose-dependent manner.0.25mmol/L concentration of IGFBP-2 MO microinjection resulted in heart malformation in nearly 60% of all injected zebrafish embryos, the abnormal phenotypes included slow heart beat, pericardial edema, weak systolic ventricle contraction, heart tube looping disorder; some of them represented atria dilation, blood regurgitation and ciculation obstruction.To validated the knocking-down effectiveness of IGFBP-2 MO, IGFBP-2 EGFP recombinant plasmid was microinjected solely or coinjected with IGFBP-2 MO and Con-MO, respectively.Wt zebrafish embryos that received single injection of IGFBP-2 EGFP recombinant plasmid or coinjection with Con-MO presented strong enhanced green fluorescence at 12hpf, meanwhile embryos coinjected with IGFBP-2 MO revealed that the EGFP expression was greatly attenuated. This strongly validated the gene specific knock-down effectiveness of IGFBP-2 MO.PartⅢIGFBP-2 gene MO knock-down influenced the Atrium specific gene expression, Peripheral angiogenesis & Ventricle specific green fluorescence of Vmhc-EGFP transgenic zebrafishSince IGFBP-2 gene knocking-down has great impact on the development of zebrafish embryonic cardiovascular system, we suppose that the cardiogenesis related gene and vascular development should also be interfered. In agreement with this notion, further investigation was carried on.Amhc was down-regulated at 48hpf in IGFBP-2 MO group. IGFBP-2 gene down-regulation to Vmhc-EGFP transgenic zebrafish also resulted in attenuated expression of ventricle specific enhanced green fluorescence at 48hpf. Intersegmental blood vessels of the IGFBP-2 MO group by microangiography at 60hpf demonstrated an sparsate and chaos image, suggesting IGFBP-2 also participate in normal peripheral vascular development of zebrafish embryo.更多还原