Hand2及Pten在第二心场增殖和发育中的功能研究

发布时间：2017-05-26 18:15

  本文关键词：Hand2及Pten在第二心场增殖和发育中的功能研究，由笔耕文化传播整理发布。

【摘要】：心血管疾病在发展中国家以及发达国家中是导致死亡的主要疾病。而在这类疾病当中,先天性心脏病,由于在胚胎发育过程中心脏发育的畸形,约影响超过5%的新生儿童的健康。因而,了解早期心脏发育的调节机制是必需的。早期心脏的前体细胞来源于侧板中胚层并由两部分前体细胞群构成,即第一心场和第二心场细胞来源。 第一心场贡献于心脏左心室的结构,而第二心场贡献于心脏流出道,右心室,静脉窦以及部分左右心房的形成。在早期心脏发育过程中,FGF,BMP,Wnt等信号对于诱导心脏特异的转录因子的表达发挥着重要的作用。然而,这些信号与转录因子如何相互作用来调节心脏发生这一过程目前研究的依然不是很清楚。Hand2,与心脏的其他转录因子如工s11,Mef2c,Fgf8/10以及FoxH等一样,对于调节心脏第二心场的发育是必需的。全身敲除Hand2基因导致胚胎致死并伴有心脏环化的缺陷,因而,我们利用Mef2c-cre转基因工具小鼠特异性的在第二心场敲除Hand2基因来研究心脏发育的过程。Hand2敲除小鼠表现有右心室发育不全和流出道发育缺陷等表型,并最终死于胚胎期第13.5天,提示着Hand2在这一区域中的必要性。通过免疫荧光染色实验,我们发现在敲除Hand2小鼠心脏中,细胞增殖是减少的,而细胞凋亡和自噬是增加的,而细胞的分化未受到明显的影响。此外,ERK信号通路的活性是下调的。以此同时,参与直接调控ERK磷酸化活性的FGFs的表达量在Hand2敲除的心脏中也是下调的。近一步实验证明Fgf10是Hand2的一个直接下游靶基因。然而,我们通过删除Pten提高ERK的活性可以明显的恢复由于Hand2缺失而造成的心脏发育缺陷的表型,不论在分子水平还是在动物水平上。与此同时,与Hand2单敲小鼠相比较,Hand2和Pten双敲小鼠的寿命明显延长。在早期胚胎发育过程中,MAPK/ERK及PI3K/AKT信号通路能够直接被FGF信号所诱导,对调节细胞的存活,增殖和分化是必需的。而之前的研究表明Pten作为肿瘤抑制基因发挥其作用通过负调控PI3K/AKT及MAPK/ERK信号通路,而Pten对脊椎动物发育的调节依然不是很清楚。是否Pten删除导致的这两条信号通路的失调对早期心脏形态发生过程有重要影响,我们着重于研究该基因在心腔第二心场中的作用。组织学研究表明,删除Pten基因在该区域导致心脏的肥大,心室间隔的发育不全,以及心脏瓣膜和心肌小梁发育的缺陷,不论是在胚胎期还是在出生后小鼠心脏均发现此类表型。以此同时,敲除Pten基因引起第二心场祖细胞的过度增殖和提前分化。近一步的研究表明Pten通过Akt-FoxO介导的上调BMP信号通路来调节祖细胞的增殖。此外,我们还发现Pten正调控NOTCH信号通路来调节心肌小梁的发育。染色体重塑复合物BAF也参与Pten介导的右心室的发育,提示着Pten可能通过表观遗传来调节第二心场的发育。综上所述,我们的研究结果表明Hand2及Pten在调节第二心场祖细胞的增殖和心脏发育过程中发挥着重要的作用。
【关键词】：Hand2 Pten 心脏发育 心脏发育缺陷 第二心场 祖细胞增殖 提前分化
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q344
【目录】：

• Abstract7-10
• 中文摘要10-12
• List of Abbreviations12-14
• Chapter 1 Introduction14-64
• 1.1. Cardiac development and signaling transduction15-33
• 1.1.1. Cardiac development from cardiac mesoderm15-17
• 1.1.2. Morphogenesis of the cardiac17-20
• 1.1.3. First heart field and second heart field20-22
• 1.1.4. Signaling pathways and transcriptional regulators in cardiac development22-33
• 1.1.4.1 Early signals in cardiac development22-26
• 1.1.4.2. Transcription factors mark cardiac progenitor cells26
• 1.1.4.3. Transcription factors in 1~(st) heart field derivatives26-28
• 1.1.4.4. Transcription factors in 2~(st) heart field derivatives28-33
• 1.2. The role of Hand transcription factors in cardiac morphogenesis33-40
• 1.2.1. Basic Helix-Loop-Helix (bHLH) Transcription Factor33-34
• 1.2.2. Hand Transcription Factors and Cardiovascular Development34-40
• 1.2.2.1. Expression of Hand transcription factors in cardiac development34-35
• 1.2.2.2. Function of Hand transcription factors in cardiac development35-38
• 1.2.2.3. Molecular and cellular mechaniams regulated by Hand238-39
• 1.2.2.4. HAND factors in human congenital heart disease (CHD)39-40
• 1.3. Characterization of Pten and its functions in development40-47
• 1.3.1. The discovery of Pten40-41
• 1.3.2. Structure, function and regulation of Pten41-45
• 1.3.2.1. Structure of Pten41-42
• 1.3.2.2 Function of Pten42
• 1.3.2.3. Regulation of Pten42-45
• 1.3.3. Function of Pten in tumorigenesis and embryonic development45-47
• 1.4. Aim and objectives47-49
• 1.5. References49-64
• Chapter 2 The HAND2-FGF-ERK Signaling Controls Proliferation of Second HeartField Progenitor64-94
• 2.1. Introduction65-67
• 2.2. Results67-85
• 2.2.1. Loss of Hand2 resulted in abnormal cardiac morphology67-69
• 2.2.1.1. Generation of Hend2 second heart field-specific knockout mice67
• 2.2.1.2. Ablation of Hand2 resulted in severe cardiac defects67-69
• 2.2.2. Cell proliferation,differentiation,apoptosis and autophagy in Hand2 mutant mice69-71
• 2.2.2.1. Ablation of Hand2 decreased the SHF progenitors and increased myocardial cells apoptosis and autophagy69-71
• 2.2.2.2. Ablation of Hand2 did not affect cell differentiation in the SHF71
• 2.2.3. Molecular studies of Hand2 mutant embryos71-75
• 2.2.3.1. Hand2 mediated activation of FGF-ERK signaling in the SHF development71-73
• 2.2.3.2. The Fgf10 promoter was activated directly by Hand273-75
• 2.2.4. Enhancement of phspho-ERK activity by loss of Pten could rescue the defects of cell proliferation in Hand2 mutant embryos75-85
• 2.2.4.1. Ablation of Pten ed to enlarged right ventricle and lengthen outflow tract75-77
• 2.2.4.2. Increased cell proliferation in Pten mulant embryos77-78
• 2.2.4.3. Ablation of Pten ed to premature differentiation of SHF progenitor cells78-80
• 2.2.4.4. Increased both of phspho-ERK and phspho-AKT activity in Pten mutant embryos80-81
• 2.2.4.5. Loss of Pten in the SHF rescued the SHF defects of Hand2 Mutants at molecular level81-83
• 2.2.4.6. Genetic ablation of Hand2 and Pten partially rescued the SHF phenotypes of Hand2 deletion mice83-85
• 2.3. Discussion85-88
• 2.4. Materials and methods88-90
• 2.5. References90-94
• Chapter 3 Pten Function in Second Heart Field is Required for Embryonic andPostnatal Heart Development94-131
• 3.1. Introduction95-96
• 3.2. Results96-118
• 3.2.1. Pten is required for embryonic cardiac development96-98
• 3.2.2. Analysis of cardiac defects in the surviving Pten~(f/f);Mef2c-Cre mice98-105
• 3.2.2.1. Loss of Pten in the second heart field results in cardiac hypertrophy and ventricular septal hyperplasia98-100
• 3.2.2.2. Loss of Pten in the second heart field results in cardiac valve and trabecular defects100-103
• 3.2.2.3. cell morphology abnormal in Pten~(f/f);Mef2c-Cre heart103-105
• 3.2.3. Pten promotes SHF cell proloferation through positvely regulating BMP signaling105-109
• 3.2.3.1. BMP signaling pathway is downregulated in Pten~(f/f);Mef2c-Cre heart105-106
• 3.2.3.2.Bmp7 promoter is directly regulated by Foxo3a in vitro106-109
• 3.2.4. Genetic ablation of Pten and Akt1 rescue RV hypertrophy and extend life span of Pten~(f/f);Mef2c-Cre mice109-110
• 3.2.5. The Combinatorial deletion of Pten and β-catenin partially rescued Pten mutant heart110-112
• 3.2.6. Analysis of Pten~(f/f);mef2c-Cre heart at late stage112-114
• 3.2.7. Chromstin-Remodeling Factors are involved in Pten~(f/f);Mef2c-Cre mice114-118
• 3.3. Discussion118-121
• 3.4. Materials and methods121-126
• 3.4.1. Animal studies121-124
• 3.4.2. Molecular studies124-126
• 3.5. References126-131
• Curriculum Vitae:Wen Luo131-133
• 致谢133-134

  本文关键词：Hand2及Pten在第二心场增殖和发育中的功能研究，由笔耕文化传播整理发布。

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