Cloning of SDMF and role in vivo

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1. Purification of smooth muscle cell derived migration factor (SDMF) : SDMF was purified from 100 L of conditioned medium of rat aortic smooth muscle cells as previously described.2. Cloning of SDMF cDNA : Purified SDMF was digested by an acid into two peptides, 25kD and 35kD peptides. 13 amino acid sequence was obtained from the 25kD peptide. The sequence was highly homologous to a certain mouse protein (protein X). Primers for protein X were synthesized and PCR products (cDNA) were obtained from mouse heart muscle, rat heart muscle and human placenta. Using these cDNA as probe, . cDNA of protein X was cloned from DNA libraries of rat culture smooth muscle cells and human placenta. Amino acid sequence derived from these cDNA contained the sequence mentioned above. cDNA from smooth muscle cells was transfected into COSl cells with an expression vector, pGM,and the protein with similar size of purified SDMF was found in the culture medium. The precise analysis of this protein is going on.3. Regulation of SDMF : TGF-beta inhibited the synthesis/secretion of SDGF from rat smooth muscle cells, being 50% inhibition at 1ng/ml. Moreover, smooth muscle cells from atheroscerotic intima secreted more SDMF than those from normal media.4. The study suggested that SDMF plays a role in the development of atherosclerosis but not in its initiation.