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

点击翻译按钮获取中文摘要

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.