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Cloning and characteristics of the novel H8D8 protein regulating valvuloseptal endocardial cushion tissue formation

调节瓣膜间隔心内膜垫组织形成的新型H8D8蛋白的克隆及特性

基本信息

批准号：
14570023
负责人：
NAKAJIMA Yuji
金额：
$ 2.24万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570023/
关键词：
CARDIAC MORPHOGENESI ENDOCARDIAL CUSHION TISSUE INDUCTIVE MOLECULE H8D8 PROTEIN 心内膜床

项目摘要

During the early heart development endothelial cells transform into mesenchyme to form valvuloseptal endocardial cushion tissue. In this endothelial-mesenchymal transformation (EMT), endothelial cells are activated by several signals, including BMP and unknown signaling(s) from the adjacent myocardium. To identify the unknown novel signaling molecule(s), which is emitted from the myocardium, we constructed a novel monoclonal antibody H8D8 against antigens that had been obtained from the concentrated myocardial conditioned medium possessing biological activities to induce the EMT. First, we established the immunohistochemical method to identify the H8D8 protein that is expressed in the cultured embryonic myocardium. We made cDNA library form the embryonic chick heart, divided them several groups containing approximately 150 cDNA clones, and transfected the group into COS cell. Resulting COS cells were examined if they were expressing H8D8 protein by means of immunohistochemistry. To dat … More e, we checked more than 10,000 clines but unfortunately we do not get H8D8-positive clone. We also tempted to identify the H8D8 protein by means of proteome analysis. We first separated the concentrated proteins obtained from cultured embryonic cardiomyocytes that are expressing H8D8 protein by 2D-gelelectrophoresis. Cell lysates of the cultured cardiomyocytes were separated two dimensionally, separated proteins were transferred to the membrane and stained by H8D8 antibody. At this time we did not obtained H8D8-positive spot in this 2D-immunoblot analysis. Further experiments are necessary to identify the H8D8 antigen. We also examined the downstream region of the BMP signaling during the EMT and clarified that both Msx1 and intracellular signaling cascade of Rho-ROCK pathway are important in the regulation of EMT. We further examined the developmental process of coronary artery stem that is established in the conotruncal cushion regions and showed several anlagen of the coronary stems develops, subsequently they fuse each other and establish coronary artery stem. Less

在心脏发育早期，内皮细胞转化为间充质，形成瓣膜间隔心内膜垫组织。在这种内皮-间充质转化过程中，内皮细胞被多种信号激活，包括骨形态发生蛋白和来自邻近心肌的未知信号(S)。为了鉴定从心肌中释放的未知新的信号分子(S)，我们构建了一种新的针对从浓缩心肌条件培养液中获得的具有诱导EMT生物活性的抗原的单抗H8D8。首先，我们建立了免疫组织化学方法来鉴定培养的胚胎心肌中表达的H8D8蛋白。我们从鸡胚胎心脏组织中构建了cDNA文库，将其分为几组，每组包含约150个克隆，并将其导入COS细胞。用免疫组织化学方法检测COS细胞是否表达H8D8蛋白。至Dat…更多，我们检查了10,000多个克隆，但不幸的是我们没有得到H8D8阳性克隆。我们还尝试通过蛋白质组分析的方法来鉴定H8D8蛋白。我们首先用2D-凝胶电泳法从培养的表达H8D8蛋白的胚胎心肌细胞中分离得到浓缩蛋白。对培养的心肌细胞裂解产物进行二维分离，将分离出的蛋白转移到细胞膜上，进行H8D8抗体染色。此时，我们在2D-免疫印迹分析中没有获得H8D8阳性斑点。需要进一步的实验来鉴定H8D8抗原。我们还研究了在EMT过程中BMP信号的下游区域，并阐明Msx1和Rho-ROCK通路的细胞内信号级联在EMT的调控中都是重要的。我们进一步观察了在圆锥干垫区建立的冠状动脉干的发育过程，发现冠状动脉干的几个原纤维发育，随后它们相互融合，形成冠状动脉干。较少