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

在心脏早期发育过程中，内皮细胞转化为间质，形成瓣膜间隔心内膜缓冲组织。在这种内皮-间充质转化（EMT）中，内皮细胞被几种信号激活，包括BMP和来自邻近心肌的未知信号。为了鉴定从心肌中释放的未知的新信号分子，我们构建了一种新的单克隆抗体H8D8，用于对抗从具有生物活性的浓缩心肌条件培养基中获得的抗原，以诱导EMT。首先，我们建立了免疫组织化学方法鉴定培养的胚胎心肌中表达的H8D8蛋白。我们从鸡胚心脏中提取cDNA文库，将其分成若干组，共含有约150个cDNA克隆，并将其转染到COS细胞中。免疫组化方法检测COS细胞是否表达H8D8蛋白。到目前为止，我们检查了1万多例，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来鉴定H8D8蛋白。我们首先通过2d凝胶电泳分离了从培养的表达H8D8蛋白的胚胎心肌细胞中获得的浓缩蛋白。将培养的心肌细胞裂解液进行二维分离，将分离的蛋白转移到膜上，用H8D8抗体进行染色。此时，我们在2d免疫印迹分析中没有获得h8d8阳性斑点。H8D8抗原的鉴定需要进一步的实验。我们还研究了EMT过程中BMP信号的下游区域，明确了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。我们进一步研究了冠状动脉干的发育过程，发现冠状动脉干的几个原细胞发育，然后相互融合，形成冠状动脉干。少

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