Purification and cloning of novel inductive factors that regulate endothelial-mesenchymal transformation in endocardial cushion tissue formation

调节心内膜垫组织形成中内皮-间质转化的新型诱导因子的纯化和克隆

• 批准号: 10670027
• 负责人: NAKAJIMA Yuji
• 金额: $ 2.5万
• 依托单位: Osaka City University, Graduate School of Medicine (2001)Saitama Medical University (1998-2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670027/
• 关键词: heart morphogenesis; endocardial cushion tissue; epithelial-mesenchymal interaction; embryonic induction

Endothelial-mesenchymal transposition is an important event that regulates the formation of the valvulo-septal endocardial cushion tissue in the early heart development. This endothelial-mesenchymal transformation is regulated by unknown signal(s) that is secreted from the myocardium of outflow tract and atrioventricular canal regions. The purpose of the present project is to elucidate the novel inductive molecule(s) that induces endothelial-mesenchymal transformation. At first, we attempted to purify the inductive molecule from embryonic myocardial conditioned medium by using different types of separation columns. Utilizing P11, hydroxyapatite, heparin sepharose, and gelatin sepharose, we obtained several fractions possessing inductive ability for endothelial-mesenchymal transformation in cultured atrioventricular endocardium on three-dimensional collagen gel (bioassay). However, we could not obtain the fractions that have inductive ability by 3-step purification processes with different columns. We next attempted to make monoclonal antibodies that recognize inductive molecule(s). We immunized rats (or mice) with crude antigens obtained from myocardial conditioned medium. Clone H8D8 recognized 70kD band in Western blot and inhibited endothelial-mesenchymal transformation in three-dimensional collagen gel assay. Immunohistochemistry showed that an H8D8-immunoreactivity was observed in cardiac jelly of stage 14-15 atrioventricular region. We next attempted to pufify the H8D8 antigen from the myocardial conditioned medium by H8D8-immuno-affinity column. We obtained a single band of H8D8 product in SDS-PAGE at molecular weight of 70KD. However, we were not able to define the partial aminoacid sequence of H8D8 protein. Finally, we made cDNA library of stage 14-16 embryonic heart. We are going to transfect them into cultured animal cells or in vitro translation system and screen the expressed proteins by using H8D8 antibody.

内皮-间充质转位是心脏早期发育过程中调节瓣膜-间隔心内膜缓冲组织形成的重要事件。这种内皮-间质转化是由流出道和房室管区心肌分泌的未知信号调控的。本项目的目的是阐明诱导内皮-间充质转化的新型诱导分子。首先，我们尝试用不同类型的分离柱从胚胎心肌条件培养基中纯化诱导分子。利用P11、羟基磷灰石、肝素蔗糖和明胶蔗糖，在三维胶原凝胶（生物测定）上获得了几种具有诱导培养房室心内膜内皮-间质转化能力的组分。但采用不同色谱柱的三步纯化工艺均不能得到具有归纳能力的馏分。接下来，我们尝试制作识别诱导分子的单克隆抗体。我们用从心肌条件培养基中获得的粗抗原免疫大鼠（或小鼠）。克隆H8D8在Western blot中识别70kD条带，在三维胶原凝胶实验中抑制内皮-间质转化。免疫组化结果显示，14-15期房室区心果冻中存在h8d8免疫反应性。接下来我们尝试用H8D8-免疫亲和柱从心肌条件培养基中纯化H8D8抗原。我们在SDS-PAGE中获得了分子量为70KD的H8D8产物单条带。然而，我们无法确定H8D8蛋白的部分氨基酸序列。最后建立了14-16期胚胎心脏cDNA文库。我们将把它们转染到培养的动物细胞或体外翻译系统中，用H8D8抗体筛选表达蛋白。