Cloning of SDMF and role in vivo

1.血管细胞源性迁移因子的纯化：从L培养的大鼠主动脉平滑肌细胞条件培养液中分离纯化出血管源性迁移因子。SDMF基因的克隆：将纯化的SDMF用酸消化，得到25kD和35kD两个多肽。从25kD的多肽中获得了13个氨基酸序列。该序列与某一种小鼠蛋白(蛋白X)高度同源。以小鼠心肌、大鼠心肌和人胎盘为模板，合成蛋白X的引物，扩增产物(CDNADNA)。以这些cDNAs为探针，获得了较好的效果。从大鼠培养的血管平滑肌细胞和人胎盘的DNA文库中克隆了蛋白X的基因。由这些cDNA推导出的氨基酸序列包含上述序列。用表达载体pGM将血管平滑肌细胞的cDNA导入COSL细胞，在培养上清液中可检测到与纯化的SDMF大小相近的蛋白。对这种蛋白质的精确分析正在进行中。血管内皮生长因子的调节：转化生长因子-β抑制大鼠血管内膜细胞合成分泌血管生长因子，1 ng/ml时抑制50%，而且动脉粥样硬化内膜的血管内膜细胞分泌的血管内皮生长因子明显高于正常对照组。这项研究表明，SDMF在动脉粥样硬化的发展过程中发挥了作用，但在其启动过程中并不起作用。

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.