小鼠心脏神经嵴细胞的生物学特性及其相关基因研究

发布时间：2018-01-12 18:35

本文关键词：小鼠心脏神经嵴细胞的生物学特性及其相关基因研究　出处：《复旦大学》2006年博士论文　论文类型：学位论文

【摘要】：圆锥动脉干是在心脏胚胎发育时期连接动脉弓的动脉干和连接心室的圆锥部的总称。心脏圆锥动脉干缺损是圆锥动脉干发育障碍所造成的一类先天性心脏病，包括法洛四联症、大动脉转位、右室双出口、永存动脉干等多种导致紫绀和低氧血症的心脏复杂畸形，这类复杂心血管畸形约占先天性心脏病的30％。人类CTD的发病机制复杂，但近二十年来的研究发现提示，Connexin 43(Cx43)基因及心脏神经嵴细胞与心脏锥干部发育密切相关。心脏神经嵴细胞是从枕部神经嵴分化出来的一群细胞，这群细胞通过第3，4，6咽弓向原始心管迁移，最后停留在动脉干和动脉圆锥等部位分化为间充质细胞，主要参与心脏流出道隔及大血管的形成。研究发现，如果在迁移之前切除散发到第3、4、6咽弓的神经嵴细胞，就会出现永存动脉干、主动脉骑跨、右室双出口、主动脉弓畸型和室间隔缺损等心血管畸形，且切除的长度与畸形的种类相关，如切除长度大于两个体节可产生永存动脉干，切除长度小于两个体节则产生右室双流出道，由此可见，心脏神经嵴细胞对于心脏圆锥部的发育意义重大。Cx43是细胞缝隙连接基因家族的成员之一，近年的研究发现其与心血管发育密切相关。研究发现，Cx43基因剔除小鼠、过量表达Cx43的CMV43转基因小鼠和显性失活抑制Cx43组成的细胞间隙的信息交流的FC转基因小鼠均可见右室流出道畸形和梗阻，从而说明，精确的Cx43基因表达对于圆锥部的发育至关重要。在小鼠胚胎发育过程中，Cx43大量表达于迁移性神经嵴细胞之上(最早于ED9.5d即可见)，并参与形成心脏神经嵴细胞的缝隙连接。越来越多的研究提示Cx43可能通过调节心脏神经嵴的行为而间接影响心脏的形态发生。因此，本课题利用Cx43基因剔除(knockout，KO)小鼠模型结合日益发展的基因芯片技术进行研究，旨在研究Cx43基因对心脏神经嵴的诱导、发生、分离、迁移、分化的调控机制，进一步阐明Cx43影响心脏圆锥发育过程其可能的分子机制。第一部分小鼠心脏神经嵴细胞的体外培养与生物学特性研究研究目的体外培养和鉴定心脏神经嵴细胞，并探讨其增殖、迁移及分化等生物学特性。材料与方法成年2月龄C57／BL6小鼠交配，取ED8.5d小鼠胚胎枕中部至第3体节神经管，应用包含碱性纤维生长因子(bEGF)和表皮细胞生长因子(EGF)的无血清培养基培养，利用MTT法检测细胞增殖能力，利用流式细胞仪检测细胞周期，采用转录激活因子2α(AP-2α，Activator protein-2α)作为其生物学标记物，观察其增殖状况、细胞周期、迁移、分化等生物学特性。结果从胎鼠神经管中分离培养的细胞增殖旺盛，AP-2α表达阳性，细胞具有明显迁移特性，传代后以含血清培养基培养后能诱导分化成神经元和神经胶质细胞。小结 1．采用无血清培养技术在体外培养可成功获得心脏神经嵴细胞； 2．体外培养的心脏神经嵴细胞增殖旺盛，具有迁移特性和多向潜能分化能力第二部分 Cx43基因敲除鼠心脏神经嵴细胞的基因表达及验证研究目的研究Cx43基因敲除(Cx43 KO)小鼠胚胎心脏神经嵴细胞中基因表达谱的改变，筛选可能导致Cx43KO小鼠圆锥动脉干畸形的相关基因，，并探讨相关机制。材料与方法以胎龄(embryonic day，ED)8.5天的Cx43KO和野生型(Cx43WT)鼠胚心脏神经嵴细胞为研究对象，分别提取总RNA，逆转录成cDNA；并在体外转录为cRNA，同时进行生物素标记及片段化；再与Affymetrix-430 2.0基因芯片进行杂交。杂交信号经扫描后，应用相关生物信息软件分析基因表达情况。选取部分差异基因应用RT-PCR方法验证基因芯片结果的可靠性。结果 1、与WT组相比，Cx43KO组中表达上调2倍以上的基因共有288个，表达下调2倍以上的基因有124个。这些差异基因主要涉及转录调控、细胞运动、细胞周期及代谢等多种功能，其中与转录调控相关的基因数改变最多。 2、PT-PCR结果与基因芯片结果相符，Affymetrix基因芯片结果能够很好的反映Cx43敲除鼠心脏神经嵴细胞与野生型鼠心脏神经嵴细胞的差异基因表达。小结利用基因芯片技术初步筛选出Cx43KO鼠心脏神经嵴细胞中多个差异基因，这些差异基因主要涉及转录调控、细胞运动、细胞周期及代谢等多种功能，其中转录调控相关基因数改变最多，提示Cx43可能主要通过转录调控等途径影响心脏神经嵴细胞的诱导、发生、分离、迁移等生物学功能并最终影响心脏圆锥部的发育。
[Abstract]:Conotruncus is in general heart during embryonic development of arterial arch artery connection and connection ventricle conus. A kind of congenital heart disease is a defect of conotruncal development disorder caused by the conotruncal heart, including tetralogy of Fallot, transposition of the great arteries, double outlet of right ventricle, truncus artery etc. leading to cyanosis and hypoxemia in complex heart malformations, the pathogenesis of complex cardiovascular malformations of congenital heart disease 30%. human CTD complex, but nearly twenty years of research found that Connexin 43 (Cx43) gene and cardiac neural crest cells closely related to conotruncal development. Cardiac neural crest cells is a group of cells from the occipital neural crest differentiation, these cells through the 3,4,6 pharyngeal arch to original heart tube migration, the last stop in the differentiation of artery and conotruncal region for mesenchymal cells, mainly Participate in the formation of cardiac outflow tract septum and blood vessel. The study found that if the neural crest cells before migrating to 3,4,6 with resection of the pharyngeal arch, there will be persistent truncus arteriosus, aorta, double outlet right ventricle, aortic arch cardiovascular ventricular septal defect and deformity, and resection length associated with the type of deformity, such as the removal of length greater than two somites can produce persistent truncus arteriosus, resection length less than two segments had double outlet of right ventricle, thus, cardiac neural crest cells is a member of the cell gap junction gene family for the development of heart in conus major.Cx43, recent studies have found that they are closely related and cardiovascular development. The study found that Cx43 gene knockout mice, FC transgenic mice can exchange information over expression of intercellular Cx43 CMV43 transgenic mice and dominant inhibition of Cx43 composed of the See the right ventricular outflow tract malformation and obstruction, and that the Cx43 gene is essential for the accurate expression of conotruncal development. During mouse embryonic development, Cx43 expression on migration of neural crest cells (the earliest in ED9.5d is visible, and participate in the formation of cracks) of CNCCs connection. More and more research suggested that Cx43 may regulate the cardiac neural crest behavior indirectly affect cardiac morphogenesis. Therefore, the issue of the use of Cx43 (knockout, KO) gene knockout mouse model with gene chip technology development of the research is to study Cx43 gene induction on cardiac neural crest, separation, transfer, regulation mechanism differentiation, further clarify the development process of Cx43 heart cone affect its possible molecular mechanism.
In vitro culture and biological characteristics of cardiac neural crest cells in part 1 of mice
research objective
In vitro culture and identification of cardiac neural crest cells, and their biological characteristics, such as proliferation, migration and differentiation, are discussed.
Materials and methods
Adult C57 / BL6 mice mating age of February, from ED8.5d to third middle occipital somite mouse embryonic neural tube, including application of basic fibroblast growth factor (bEGF) and epidermal growth factor (EGF) in serum-free culture medium, detection of cell proliferation by MTT assay, the cell cycle was analyzed by flow cytometry, the transcription the activation of factor 2 alpha (AP-2 alpha, alpha Activator protein-2) as the biological marker to observe the proliferation, cell cycle, migration, differentiation and other biological characteristics.
Result
The proliferation and proliferation of cells from fetal rat neural tube were strong. The expression of AP-2 alpha was positive, and the cells had obvious migration characteristics. After passage, cultured in serum containing medium, they could be induced to differentiate into neurons and glial cells.
Summary
1. the heart neural crest cells can be successfully obtained by serum-free culture in vitro.
2. in vitro cultured cardiac neural crest cells proliferate vigorously, and have the ability of migration and multipotential differentiation.
Gene expression and verification of the second part of Cx43 gene knockout rat heart neural crest cells
research objective
Objective to study the changes of gene expression profiles in embryonic cardiac neural crest cells of Cx43 knockout (Cx43 KO) mice, and screen related genes that may cause cones arteriosus malformation in Cx43KO mice, and explore the related mechanisms.
Materials and methods
For gestational age (embryonic day, ED) 8.5 days Cx43KO and wild type (Cx43WT) of rat embryonic cardiac neural crest cells as the object of study, total RNA was extracted, reverse transcribed into cDNA; and in vitro transcribed into cRNA, and biotin labeling and fragmentation; and Affymetrix-430 2 gene chip hybridization. By scanning the hybridization signal, analysis of gene expression related to application of bioinformatics software. The reliability of selected genes using RT-PCR method to verify the results of gene chip.
Result
1, compared with the WT group, Cx43KO group, expression of 2 times more than 288 genes were downregulated, 2 times more than 124 genes. These genes were mainly involved in transcriptional regulation, cell movement, cell cycle and metabolism function, and transcriptional regulation of genes related to the change in the number of the most.
2, the PT-PCR results were consistent with the results of gene chip. Affymetrix microarray results could well reflect the differential gene expression between cardiac Cx43 and knockout rat cardiac neural crest cells.
Summary
Using the gene chip technology was screened multiple gene differences in Cx43KO rat cardiac neural crest cells, these genes were mainly involved in regulation of transcription, cell movement, cell cycle and metabolism of a variety of functions, including transcriptional regulation of genes related to change the number of the most, suggesting that Cx43 may be mainly through transcriptional regulation pathways induced cardiac neural crest cells the occurrence of separation, migration and other biological functions and ultimately affect cardiac conotruncal development.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R725.4;R329

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