蛋白質香葉基香葉基化修飾在小鼠胚胎期心臟發(fā)育中的功能研究
發(fā)布時間：2020-10-17 18:40
先天性心臟病(CHD)是一種常見的出生缺陷。原衛(wèi)生部于2012年發(fā)布的《中國出生缺陷防治報告》指出我國先天性心臟病的發(fā)病率在2000-2011年間呈上升趨勢,到2011年其病例已占所有圍產(chǎn)期出生缺陷病例的26.7%。由此可見,先天性心臟病仍舊是一個亟待解決的公共衛(wèi)生問題。胚胎期心臟的發(fā)育異常是引起先天性心臟病的主要原因,因此研究早期心臟發(fā)育的調(diào)控機制對于深入了解先天性心臟病的發(fā)病機理以及開發(fā)更有效的治療策略具有重要的意義。甲羥戊酸途徑是細胞中合成甾醇和非甾體類異戊二烯的重要代謝途徑。其下游的兩個非甾體類異戊二烯產(chǎn)物法尼基二磷酸(FPP)和香葉基香葉基二磷酸(GGPP)是參與蛋白質異戊二烯化修飾的重要底物。蛋白質異戊二烯化修飾作為一種蛋白質翻譯后修飾參與眾多細胞生理過程。已有研究表明甲羥戊酸途徑上游HMG-CoA還原酶以及下游香葉基香葉基焦磷酸合成酶(Ggpps,負責將FPP轉化為GGPP)的突變,亦或藥理性抑制異戊二烯基轉移酶活性可阻礙果蠅和斑馬魚心管的形成。此外,我們實驗室先前的研究結果表明蛋白質異戊二烯化修飾參與小鼠出生后的心肌肥大性生長。這些結果提示蛋白質異戊二烯化修飾與心臟發(fā)育之間存在密切的聯(lián)系。然而蛋白質異戊二烯化修飾在哺乳動物胚胎期心臟發(fā)育過程中發(fā)揮怎樣的作用仍未有相關研究闡明。首先,我們發(fā)現(xiàn)GGPPS在小鼠心臟發(fā)育過程中持續(xù)表達,并且其mRNA和蛋白表達水平在胚胎期10.5天(E10.5)后顯著升高,這提示GGPPS調(diào)控的蛋白質異戊二烯化修飾可能在小鼠胚胎發(fā)育中期的心臟發(fā)育過程中發(fā)揮重要作用。為進一步證實這種潛在的作用,我們利用Nkx2.5Cre/+和α-SMA-Cre工具鼠與Ggppsfl/fl鼠交配構建了兩種在不同發(fā)育時期心臟特異性缺失Ggpps的動物模型。我們發(fā)現(xiàn)利用Nkx2.5Cre/+在心臟祖細胞中敲除Ggpps會從E10.5起破壞心肌細胞中的蛋白質香葉基香葉基化修飾并造成突變體胚胎在E12.0到E13.0間死亡。組織形態(tài)學觀察發(fā)現(xiàn),從E11.5天起,Nkx2.5Cre/+,;Ggppsfl/fl突變胚胎的心臟與對照組相比逐漸變小并伴有心室腔發(fā)育的異常,表現(xiàn)為心室間隔以及心肌小梁的生長阻滯。免疫熒光染色的結果顯示,從E11.5天起,Nkx2.5Cre/+;Ggppsfl/fl突變體心臟中細胞增殖水平逐漸降低,然而細胞凋亡卻并未有明顯改變。因此心肌細胞增殖的減弱可能是造成突變體心臟變小以及心室腔發(fā)育異常的重要原因。我們通過免疫組化染色還發(fā)現(xiàn),在心肌小梁中特異性表達的蛋白ANF在Nkx2.5Cre/+;Ggppsfl/fl突變體的心室致密層中出現(xiàn)了異位表達的現(xiàn)象。與此同時,心室致密層的標志蛋白Hey2在突變體心臟的致密層中幾乎消失了。這種心臟中區(qū)域特異性基因表達的異常也可能通過破壞致密層心肌細胞的分化狀態(tài)參與心室腔的異常發(fā)育。除此以外,我們還發(fā)現(xiàn)Nkx2.5Cre/+;Ggppsfl/fl突變體心臟中心外膜的發(fā)育也出現(xiàn)了異常,主要表現(xiàn)為心外膜來源細胞發(fā)生過度的EMT以及增殖的現(xiàn)象。當我們運用α-SMA-Cre在已分化的心肌細胞中敲除Ggpps時,心肌細胞中的蛋白質香葉基香葉基化修飾從E13.5起被明顯抑制。有趣的是,α-SMA-Cre;Ggppsfl/fl突變體胚胎可以存活到出生并且其胚胎期的心臟發(fā)育并未有明顯變化。以上結果提示小鼠胚胎心臟發(fā)育可能階段依賴性地需要GGPPS所調(diào)控的蛋白質香葉基香葉基化修飾。而這個重要的階段可能介于E10.5到E13.5之間。由于在Nkx2.5Cre/+;Ggppsfl/fl突變體心臟中嚴重的缺陷主要發(fā)生在E12.5,而此時的突變體胚胎已經(jīng)瀕臨死亡。再者從E11.5起才出現(xiàn)的心室腔發(fā)育異常,如心室間隔及心肌小梁的生長阻滯,心外膜發(fā)育的異常等并不足以使得突變體胚胎在E12.0至E13.0間快速死亡。因此我們進一步分析了Nkx2.5Cre/+;Ggppsfl/fl突變體心臟在E12.5之前可能存在的其他異常。值得注意的是心臟中心肌細胞的有序構筑是維持心臟正常收縮功能以及促進心室腔成熟過程中形態(tài)重構的重要因素。我們利用透射電子顯微鏡觀察發(fā)現(xiàn)在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突變體心臟中心肌細胞的肌節(jié)松弛,細胞與細胞間的連接結構稀疏。我們將此時期的突變體心肌細胞分離后進行體外培養(yǎng),發(fā)現(xiàn)其細胞貼壁能力降低并且殘存的貼壁細胞無法充分鋪展。進一步利用免疫熒光染色檢測細胞黏著連接蛋白N-cadherin和β-catenin的細胞內(nèi)定位,結果發(fā)現(xiàn)二者在E10.5的突變體心臟中共定位在相鄰細胞連接處,相比于對照心臟并未有明顯差異。但是在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突變體心臟中N-cadherin和β-catenin喪失了在細胞連接處的共定位而彌散分布在細胞質中,并且通過免疫共沉淀分析我們也發(fā)現(xiàn)二者在突變體心臟中的相互作用也減弱了。此外我們還發(fā)現(xiàn)介導細胞間隙連接的蛋白Connexin43的細胞膜定位也在E11.5的Nkx2.5Cre/+;Ggppsfl/fl突變體心肌細胞中喪失了。以上結果提示由Nkx2.5Cre/+介導的心肌細胞Ggpps敲除早在E11.5就破壞了心肌細胞之間的連接并造成突變體心臟中心肌細胞構筑的紊亂,這可能是造成突變體胚胎在E12.0至E13.0間快速死亡的重要原因。受蛋白質香葉基香葉基化修飾的小G蛋白,如Rho GTPase家族,參與對細胞骨架及細胞連接的調(diào)控。我們在Nkx25Cre/+,;Ggppsfl/fl突變體心臟中發(fā)現(xiàn)Rho GTPases家族中的兩個重要成員RhoA和Racl的膜定位,異戊二烯化修飾水平以及GTPase的酶活性均較對照心臟有明顯降低。并且在體內(nèi)和體外水平上外源補充GGPP提高小G蛋白的香葉基香葉基化水平能在一定程度上恢復Nkx2.5Cre/+;Ggppsfl/fl突變體心肌細胞連接以及細胞構筑的異常。以上結果提示由Ggpps缺失引起的心肌細胞構筑的紊亂可能是由諸如Rho GTPase等受香葉基香葉基化修飾的小G蛋白活性降低引起的。綜上所述,我們的研究結果表明GGPPS調(diào)控的蛋白質香葉基香葉基化修飾作為一種階段特異的信號促進胚胎發(fā)育中期心肌細胞在心臟中有序構筑,進而維持了心室腔的正常發(fā)育。
【學位單位】：南京大學
【學位級別】：博士
【學位年份】：2018
【中圖分類】：R541.1
【文章目錄】：
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
致謝
Publication
【參考文獻】
本文編號：2845175
【學位單位】：南京大學
【學位級別】：博士
【學位年份】：2018
【中圖分類】：R541.1
【文章目錄】：
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
致謝
Publication
【參考文獻】
相關期刊論文 前1條
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
本文編號：2845175
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