Cloning of SDGF gene and development of its specific inhibitors

1. 平滑肌细胞衍生生长因子（SDGF）的免疫学分析：从培养的兔主动脉平滑肌细胞中获得SDMF。抗人碱性成纤维细胞生长因子（FGF）的多克隆抗体特异性抑制SDMF活性。但SDGF的理化性质与FGF不同。兔条件培养基的western blot分析显示，SDMF的分子量为32 kD，而FGF的分子量为17-18kD。这些结果表明，SDGF是FGF超家族的成员，而不是FGF本身。SDGF cDNA的克隆：为了分析SDGF的氨基酸序列，预计需要大量的条件培养基。因此，我们首先筛选SDGF cDNA，使用特异性抗体。然而，灵敏度是不够的。接下来，我们构建了包含FGF超家族公共序列的引物。从分泌SDGF的兔平滑肌细胞中获得cDNA文库。从这些文库中获得了分子量约为300bp的PCR产物。分析正在进行中，使用其中一个cDNA将获得全长cDNA。SDGF抑制剂：许多内源性和外源性抑制剂对SDGF进行了测试。其中，tgf - β（内源性细胞因子）和钙拮抗剂effonidipin是有效的抑制剂。

1. Immunological analysis of smooth muscle cell derived growth factor (SDGF) : SDMF was obtained from cultured rabbit aortic smooth muscle cells. SDMF activity was specifically inhibited by a polyclonal antibody against human basic fibroblast growth factor (FGF). But physico-chemical properties of SDGF were different from those of FGF.Moreover, the western blot analysis of rabbit conditioned medium showed that the molecular size of SDMF was 32 kD wheras that of FGF was 17-18kD.These results indicate that SDGF was a member of FGF superfamily but not FGF itself.2. Cloning of SDGF cDNA : For the analysis of amino acid sequence of SDGF,large amount of conditioned medium was expected to be needed. Therefore, we first screened the SDGF cDNA,using the specific antibody. However, the sensitivity was not enough. Next, we made up primers containing common sequences of FGF superfamily. cDNA libraries were obtained from cultured rabbit smooth muscle cells which secreted SDGF.PCR products with a molecular size about 300bp were obtained from these libraries. The nalysis of this is going on and full length cDNA will be obtained using one of these cDNA.3. Inhibitors of SDGF : Many endogenous and exogenous inhibitors were tested against SDGF.Among them, TGF-beta (endogenous cytokine) and a calcium antagonist, effonidipin were potent inhibitors.