蛋白质香叶基香叶基化修饰在小鼠胚胎期心脏发育中的功能研究
发布时间：2020-10-17 18:40
先天性心脏病(CHD)是一种常见的出生缺陷。原卫生部于2012年发布的《中国出生缺陷防治报告》指出我国先天性心脏病的发病率在2000-2011年间呈上升趋势,到2011年其病例已占所有围产期出生缺陷病例的26.7%。由此可见,先天性心脏病仍旧是一个亟待解决的公共卫生问题。胚胎期心脏的发育异常是引起先天性心脏病的主要原因,因此研究早期心脏发育的调控机制对于深入了解先天性心脏病的发病机理以及开发更有效的治疗策略具有重要的意义。甲羟戊酸途径是细胞中合成甾醇和非甾体类异戊二烯的重要代谢途径。其下游的两个非甾体类异戊二烯产物法尼基二磷酸(FPP)和香叶基香叶基二磷酸(GGPP)是参与蛋白质异戊二烯化修饰的重要底物。蛋白质异戊二烯化修饰作为一种蛋白质翻译后修饰参与众多细胞生理过程。已有研究表明甲羟戊酸途径上游HMG-CoA还原酶以及下游香叶基香叶基焦磷酸合成酶(Ggpps,负责将FPP转化为GGPP)的突变,亦或药理性抑制异戊二烯基转移酶活性可阻碍果蝇和斑马鱼心管的形成。此外,我们实验室先前的研究结果表明蛋白质异戊二烯化修饰参与小鼠出生后的心肌肥大性生长。这些结果提示蛋白质异戊二烯化修饰与心脏发育之间存在密切的联系。然而蛋白质异戊二烯化修饰在哺乳动物胚胎期心脏发育过程中发挥怎样的作用仍未有相关研究阐明。首先,我们发现GGPPS在小鼠心脏发育过程中持续表达,并且其mRNA和蛋白表达水平在胚胎期10.5天(E10.5)后显著升高,这提示GGPPS调控的蛋白质异戊二烯化修饰可能在小鼠胚胎发育中期的心脏发育过程中发挥重要作用。为进一步证实这种潜在的作用,我们利用Nkx2.5Cre/+和α-SMA-Cre工具鼠与Ggppsfl/fl鼠交配构建了两种在不同发育时期心脏特异性缺失Ggpps的动物模型。我们发现利用Nkx2.5Cre/+在心脏祖细胞中敲除Ggpps会从E10.5起破坏心肌细胞中的蛋白质香叶基香叶基化修饰并造成突变体胚胎在E12.0到E13.0间死亡。组织形态学观察发现,从E11.5天起,Nkx2.5Cre/+,;Ggppsfl/fl突变胚胎的心脏与对照组相比逐渐变小并伴有心室腔发育的异常,表现为心室间隔以及心肌小梁的生长阻滞。免疫荧光染色的结果显示,从E11.5天起,Nkx2.5Cre/+;Ggppsfl/fl突变体心脏中细胞增殖水平逐渐降低,然而细胞凋亡却并未有明显改变。因此心肌细胞增殖的减弱可能是造成突变体心脏变小以及心室腔发育异常的重要原因。我们通过免疫组化染色还发现,在心肌小梁中特异性表达的蛋白ANF在Nkx2.5Cre/+;Ggppsfl/fl突变体的心室致密层中出现了异位表达的现象。与此同时,心室致密层的标志蛋白Hey2在突变体心脏的致密层中几乎消失了。这种心脏中区域特异性基因表达的异常也可能通过破坏致密层心肌细胞的分化状态参与心室腔的异常发育。除此以外,我们还发现Nkx2.5Cre/+;Ggppsfl/fl突变体心脏中心外膜的发育也出现了异常,主要表现为心外膜来源细胞发生过度的EMT以及增殖的现象。当我们运用α-SMA-Cre在已分化的心肌细胞中敲除Ggpps时,心肌细胞中的蛋白质香叶基香叶基化修饰从E13.5起被明显抑制。有趣的是,α-SMA-Cre;Ggppsfl/fl突变体胚胎可以存活到出生并且其胚胎期的心脏发育并未有明显变化。以上结果提示小鼠胚胎心脏发育可能阶段依赖性地需要GGPPS所调控的蛋白质香叶基香叶基化修饰。而这个重要的阶段可能介于E10.5到E13.5之间。由于在Nkx2.5Cre/+;Ggppsfl/fl突变体心脏中严重的缺陷主要发生在E12.5,而此时的突变体胚胎已经濒临死亡。再者从E11.5起才出现的心室腔发育异常,如心室间隔及心肌小梁的生长阻滞,心外膜发育的异常等并不足以使得突变体胚胎在E12.0至E13.0间快速死亡。因此我们进一步分析了Nkx2.5Cre/+;Ggppsfl/fl突变体心脏在E12.5之前可能存在的其他异常。值得注意的是心脏中心肌细胞的有序构筑是维持心脏正常收缩功能以及促进心室腔成熟过程中形态重构的重要因素。我们利用透射电子显微镜观察发现在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突变体心脏中心肌细胞的肌节松弛,细胞与细胞间的连接结构稀疏。我们将此时期的突变体心肌细胞分离后进行体外培养,发现其细胞贴壁能力降低并且残存的贴壁细胞无法充分铺展。进一步利用免疫荧光染色检测细胞黏着连接蛋白N-cadherin和β-catenin的细胞内定位,结果发现二者在E10.5的突变体心脏中共定位在相邻细胞连接处,相比于对照心脏并未有明显差异。但是在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突变体心脏中N-cadherin和β-catenin丧失了在细胞连接处的共定位而弥散分布在细胞质中,并且通过免疫共沉淀分析我们也发现二者在突变体心脏中的相互作用也减弱了。此外我们还发现介导细胞间隙连接的蛋白Connexin43的细胞膜定位也在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突变体心肌细胞中丧失了。以上结果提示由Nkx2.5Cre/+介导的心肌细胞Ggpps敲除早在E11.5就破坏了心肌细胞之间的连接并造成突变体心脏中心肌细胞构筑的紊乱,这可能是造成突变体胚胎在E12.0至E13.0间快速死亡的重要原因。受蛋白质香叶基香叶基化修饰的小G蛋白,如Rho GTPase家族,参与对细胞骨架及细胞连接的调控。我们在Nkx25Cre/+,;Ggppsfl/fl突变体心脏中发现Rho GTPases家族中的两个重要成员RhoA和Racl的膜定位,异戊二烯化修饰水平以及GTPase的酶活性均较对照心脏有明显降低。并且在体内和体外水平上外源补充GGPP提高小G蛋白的香叶基香叶基化水平能在一定程度上恢复Nkx2.5Cre/+;Ggppsfl/fl突变体心肌细胞连接以及细胞构筑的异常。以上结果提示由Ggpps缺失引起的心肌细胞构筑的紊乱可能是由诸如Rho GTPase等受香叶基香叶基化修饰的小G蛋白活性降低引起的。综上所述,我们的研究结果表明GGPPS调控的蛋白质香叶基香叶基化修饰作为一种阶段特异的信号促进胚胎发育中期心肌细胞在心脏中有序构筑,进而维持了心室腔的正常发育。
【学位单位】：南京大学
【学位级别】：博士
【学位年份】：2018
【中图分类】：R541.1
【文章目录】：
摘要
Abstract
CHAPTER Ⅰ A Brief Review: Cardiac chamber development and protein prenylation
1.1 An overview of heart organogenesis
1.2 Major cell types in the cardiac chamber
1.2.1 Myocardium
1.2.2 Endocardium
1.2.3 Epicardium
1.3 Cardiac chamber maturation
1.3.1 Cardiac trabecualtion
1.3.2 The regulation of cardiac trabeculation
1.3.3 Conduction system development
1.3.4 Myocardial proliferation in the compact layer
1.3.5 Cardiac cytoarchitecture and its role in the cardiac chamber development
1.4 Mevalonate pathway and protein prenylation
1.4.1 Mevalonate pathway
1.4.2 Protein prenylation
1.5 The role of protein prenylation in the heart diseases and development
1.6 References
CHAPTER Ⅱ The expression of Ggpps is augmented in mouse hearts during mid-gestation
2.1 Introduction
2.2 Materials and methods
2.2.1 Mice
2.2.2 Genotyping
2.2.3 Immunohistochemistry and Immunofluorescence staining
2.2.4 RNA extraction and Quantitative PCR
2.2.5 Immunoblot analysis
2.2.6 Cell culture
2.2.7 Statistics analysis
2.3 Results
2.3.1 The expression of Ggpps increased in a stage-dependent manner in the developingheart
Cre/+ and α-SMA-Cre lines'> 2.3.2 Expression pattern of Nkx2Cre/+ and α-SMA-Cre lines
2.4 Summary
2.5 Discussion
2.6 References
CHAPTER Ⅲ Protein geranylgeranylation is stage-dependently required for mammalian heart developmen
3.1 Introduction
3.2 Materials and methods
3.2.1 Mice
3.2.2 Genotyping
3.2.3 Histological analysis, immunohistochemistry and immunofluorescence staining
3.2.4 RNA extraction and Quantitative PCR
3.2.5 Immunoblot analysis
3.2.6 Tunel assay
3.2.7 Primary epicardial cells culture and immunofluorescence
3.2.8 Statistics analysis
3.3 Results
Cre/+ resulted in mid-gestational lethality'> 3.3.1 Cardiac inactivation of Ggpps by Nkx2.5Cre/+ resulted in mid-gestational lethality
3.3.2 Cardiac inactivation of Ggpps by Nkx2.5（Cre/+） disrupted heart development during mid-gestation
Cre/- arrested heart growth duringmid-gestation'> 3.3.2.1 Cardiac inactivation of Ggpps by Nkx2.5Cre/- arrested heart growth duringmid-gestation
Cre/- resulted in aberrant myocardialproliferation, but had no effects on myocardial apoptosis'> 3.3.2.2 Cardiac inactivation of Ggpps by Nkx2.5Cre/- resulted in aberrant myocardialproliferation, but had no effects on myocardial apoptosis
Cre/- resulted in the abnormaltrabecular regional specification'> 3.3.2.3 Cardiac inactivation of Ggpps by Nkx2.5Cre/- resulted in the abnormaltrabecular regional specification
Cre/- caused abnormal epicardiumdevelopment'> 3.3.2.4 Cardiac inactivation of Ggpps by Nkx2.5Cre/- caused abnormal epicardiumdevelopment
3.3.3 Later cardiac inactivation of Ggpps by α-SMA-Cre did not affect embryonic heart development
3.4 Summary
3.5 Discussion
3.6 References
CHAPTER Ⅳ Protein geranylgeranylation regulates the organization of cardiomyocytes through modulating RhoGTPase activity
4.1 Introduction
4.2 Materials and methods
4.2.1 Mice
4.2.2 Histological analysis, immunohistochemistry and immunofluorescence staining
4.2.3 WGA staining
4.2.4 Transmission electron microscopy
4.2.5 Immunoblot analysis
4.2.6 Immunoprecipitation analysis
4.2.7 RNA extraction and Quantitative PCR
4.2.8 Cell culture
4.2.9 Triton X-114 extraction of hydrophobic proteins
4.2.10 Small GTPase activity assay
4.2.11 Statistical analysis
4.3 Results
4.3.1 Mid-gestational disruption of protein geranylgeranylation led to the disorganization ofcardiac cytoarchitecture
4.3.2 Protein geranylgeranylation was essential for cell-cell junctions in cardiomyocytesduring mid-gestation
4.3.3 Mid-gestational disruption of protein geranylgeranylation disturbed cardiaccytoarchitectural establishment by inhibiting Rho GTP ases activity
4.4 Summary
4.5 Discussion
4.6 References
Appendix
致谢
Publication
【参考文献】
本文编号：2845175
【学位单位】：南京大学
【学位级别】：博士
【学位年份】：2018
【中图分类】：R541.1
【文章目录】：
摘要
Abstract
CHAPTER Ⅰ A Brief Review: Cardiac chamber development and protein prenylation
1.1 An overview of heart organogenesis
1.2 Major cell types in the cardiac chamber
1.2.1 Myocardium
1.2.2 Endocardium
1.2.3 Epicardium
1.3 Cardiac chamber maturation
1.3.1 Cardiac trabecualtion
1.3.2 The regulation of cardiac trabeculation
1.3.3 Conduction system development
1.3.4 Myocardial proliferation in the compact layer
1.3.5 Cardiac cytoarchitecture and its role in the cardiac chamber development
1.4 Mevalonate pathway and protein prenylation
1.4.1 Mevalonate pathway
1.4.2 Protein prenylation
1.5 The role of protein prenylation in the heart diseases and development
1.6 References
CHAPTER Ⅱ The expression of Ggpps is augmented in mouse hearts during mid-gestation
2.1 Introduction
2.2 Materials and methods
2.2.1 Mice
2.2.2 Genotyping
2.2.3 Immunohistochemistry and Immunofluorescence staining
2.2.4 RNA extraction and Quantitative PCR
2.2.5 Immunoblot analysis
2.2.6 Cell culture
2.2.7 Statistics analysis
2.3 Results
2.3.1 The expression of Ggpps increased in a stage-dependent manner in the developingheart
Cre/+ and α-SMA-Cre lines'> 2.3.2 Expression pattern of Nkx2Cre/+ and α-SMA-Cre lines
2.4 Summary
2.5 Discussion
2.6 References
CHAPTER Ⅲ Protein geranylgeranylation is stage-dependently required for mammalian heart developmen
3.1 Introduction
3.2 Materials and methods
3.2.1 Mice
3.2.2 Genotyping
3.2.3 Histological analysis, immunohistochemistry and immunofluorescence staining
3.2.4 RNA extraction and Quantitative PCR
3.2.5 Immunoblot analysis
3.2.6 Tunel assay
3.2.7 Primary epicardial cells culture and immunofluorescence
3.2.8 Statistics analysis
3.3 Results
Cre/+ resulted in mid-gestational lethality'> 3.3.1 Cardiac inactivation of Ggpps by Nkx2.5Cre/+ resulted in mid-gestational lethality
3.3.2 Cardiac inactivation of Ggpps by Nkx2.5（Cre/+） disrupted heart development during mid-gestation
Cre/- arrested heart growth duringmid-gestation'> 3.3.2.1 Cardiac inactivation of Ggpps by Nkx2.5Cre/- arrested heart growth duringmid-gestation
Cre/- resulted in aberrant myocardialproliferation, but had no effects on myocardial apoptosis'> 3.3.2.2 Cardiac inactivation of Ggpps by Nkx2.5Cre/- resulted in aberrant myocardialproliferation, but had no effects on myocardial apoptosis
Cre/- resulted in the abnormaltrabecular regional specification'> 3.3.2.3 Cardiac inactivation of Ggpps by Nkx2.5Cre/- resulted in the abnormaltrabecular regional specification
Cre/- caused abnormal epicardiumdevelopment'> 3.3.2.4 Cardiac inactivation of Ggpps by Nkx2.5Cre/- caused abnormal epicardiumdevelopment
3.3.3 Later cardiac inactivation of Ggpps by α-SMA-Cre did not affect embryonic heart development
3.4 Summary
3.5 Discussion
3.6 References
CHAPTER Ⅳ Protein geranylgeranylation regulates the organization of cardiomyocytes through modulating RhoGTPase activity
4.1 Introduction
4.2 Materials and methods
4.2.1 Mice
4.2.2 Histological analysis, immunohistochemistry and immunofluorescence staining
4.2.3 WGA staining
4.2.4 Transmission electron microscopy
4.2.5 Immunoblot analysis
4.2.6 Immunoprecipitation analysis
4.2.7 RNA extraction and Quantitative PCR
4.2.8 Cell culture
4.2.9 Triton X-114 extraction of hydrophobic proteins
4.2.10 Small GTPase activity assay
4.2.11 Statistical analysis
4.3 Results
4.3.1 Mid-gestational disruption of protein geranylgeranylation led to the disorganization ofcardiac cytoarchitecture
4.3.2 Protein geranylgeranylation was essential for cell-cell junctions in cardiomyocytesduring mid-gestation
4.3.3 Mid-gestational disruption of protein geranylgeranylation disturbed cardiaccytoarchitectural establishment by inhibiting Rho GTP ases activity
4.4 Summary
4.5 Discussion
4.6 References
Appendix
致谢
Publication
【参考文献】
相关期刊论文 前1条
1 XU Na;SHEN Ning;WANG XiuXing;JIANG Shan;XUE Bin;LI ChaoJun;;Protein prenylation and human diseases: a balance of protein farnesylation and geranylgeranylation[J];Science China(Life Sciences);2015年04期
本文编号：2845175
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2845175.html
最近更新
教材专著
