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Cloning of SDGF gene and development of its specific inhibitors

SDGF基因的克隆及其特异性抑制剂的开发

基本信息

批准号：
06557053
负责人：
MORISAKI Nobuhiro
金额：
$ 8.26万
依托单位：
Chiba Unibersity
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557053/
关键词：
SDGF Smoothmuscle cells Basic fibroblast growth factor Atherosclerosis Effonidipin

项目摘要

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.

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获得SDMF。SDMF的活性特异性抑制的多克隆抗体对人碱性成纤维细胞生长因子（FGF）。但SDGF的理化性质与FGF不同，而且兔条件培养液的western blot分析表明，SDGF的分子大小为32 kD，而FGF的分子大小为17- 18 kD，说明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，我们首先使用特异性抗体筛选SDGF cDNA。然而，灵敏度还不够。接下来，我们制备了含有FGF超家族共同序列的引物。从培养的分泌SDGF的兔平滑肌细胞中获得cDNA文库，并从文库中获得分子大小约为300 bp的PCR产物。目前正在进行分析，并将利用其中一种cDNA获得全长cDNA. SDGF抑制剂：许多内源性和外源性的抑制剂被测试，其中TGF-β（内源性细胞因子）和钙拮抗剂，effonidipin是有效的抑制剂。