Cloning of SDMF and role in vivo

SDMF 的克隆及其体内作用

• 批准号: 06836002
• 负责人: MORISAKI Nobuhiro
• 金额: $ 1.41万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 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/en/grant/KAKENHI-PROJECT-06836002/
• 关键词: SDMF; Smooth muscle cells; Atherosclerosis; Intimal smooth muscle cells; Trans forming growth factor-B; トランスフォーミング成長因子-β

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.

1.平滑肌细胞衍生的迁移因子（SDMF）的纯化：如前所述，从100 μ L大鼠主动脉平滑肌细胞的条件培养基中纯化SDMF. SDMF cDNA的克隆：将纯化的SDMF用酸消化成25 kD和35 kD两个肽段。从25 kD多肽中获得13个氨基酸序列。该序列与某种小鼠蛋白（蛋白X）高度同源。合成蛋白X的引物，从小鼠心肌、大鼠心肌和人胎盘中获得PCR产物（cDNA）。以这些cDNA为探针，从培养的大鼠平滑肌细胞和人胎盘DNA文库中克隆了X蛋白的cDNA。从这些cDNA衍生的氨基酸序列包含上述序列。用表达载体pGM转染COS 1细胞，在培养液中发现与纯化的SDMF大小相似的蛋白。这种蛋白质的精确分析正在进行中。SDMF的调节：TGF-β抑制大鼠平滑肌细胞SDGF的合成/分泌，在1 ng/ml时抑制率为50%。此外，动脉粥样硬化内膜平滑肌细胞分泌的SDMF也高于正常中膜平滑肌细胞.这项研究表明，SDMF在动脉粥样硬化的发展中起作用，但在其起始中不起作用。

项目成果

Morisaki N,Koyama N,Saito Y,Yoshida S: "Purification and Characterization of an Autocrine Migration Factor for Vascular Smooth Muscle Cells,and Its Role in Arteriosclerosis" Ann New York Acad Sci. 748. 575-577 (1995)

Morisaki N、Koyama N、Saito Y、Yoshida S：“血管平滑肌细胞自分泌迁移因子的纯化和表征及其在动脉硬化中的作用”Ann New York Acad Sci。

Morisaki N,Kanzaki T,Tamura K,Saito I,Shiina R,Saito Y: "Specific inhibition of vascular cell adhesion molecule-1 expression by type four collagen in endothelial cells." Biochem Biophys Res Commun. 214. 1163-1167 (1995)

Morisaki N、Kanzaki T、Tamura K、Saito I、Shiina R、Saito Y：“内皮细胞中四型胶原对血管细胞粘附分子 1 表达的特异性抑制。”

Morisaki N,Yokote K.Takahashi K,Otabe M,Saito Y,Yoshida S.Ueda S: "Role of phespholigase A_2 in expression of the scaverrger pathway in cultured aorbic smoothmuscle cells stimulated with phorbol 12-myristate 13-ocetate" Biochem J. 303. 247-253 (1994)

Morisaki N，Yokote K.Takahashi K，Otabe M，Saito Y，Yoshida S.Ueda S：“在用佛波醇 12-肉豆蔻酸酯 13-辛酸酯刺激的培养的有氧平滑肌细胞中，phespholigase A_2 在清道夫途径表达中的作用”Biochem J。

Kanzaki T,Morisaki N: "Effect of TGF-beta on arterial wall cells-in vitro, in vivo. Mebio" 11. 87-92 (1994)

Kanzaki T、Morisaki N：“TGF-β 对动脉壁细胞的影响 - 体外、体内。Mebio”11. 87-92 (1994)

Morisaki N,Koyama N,Saito Y,Yoshida S: "Purification and characterization of an anticrone migration factor for vascular smooth muscle cells and its role in anteriosclerosis" Ann New York Acad Sci. 748. 575-577 (1995)

Morisaki N、Koyama N、Saito Y、Yoshida S：“血管平滑肌细胞反克隆迁移因子的纯化和表征及其在动脉硬化中的作用”Ann New York Acad Sci。