In vivo analysis of osteopontin function

骨桥蛋白功能的体内分析

• 批准号: 08457074
• 负责人: HIGUCHI Yasunori
• 金额: $ 4.74万
• 依托单位: Oita Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457074/
• 关键词: osteopontin; transgene; metallothionein; CD14; LPS; Inflammation; オステオポンチン; トランスジエニック; リコンビナント; 炎症; がん; 接着; 抗オステオポンチン抗体; 単クローン抗体; 粥状硬化; トランスジェニックマウス

1)Production and analysis of anti-mouse osteopontin (OPN) monoclonal antibodies (mAb) : mAb, which show different features of tissue reactivity, were produced. mAb A reacts with kidney and bone cells, whereas mAb B reacts with kidney but not with bone cells. Analysis using these mAb showed that the different reactivity is ascribable to masking of epitope. Functional domains were analyzed using mAb and recombinant OPN,and found that an RGD sequence plays a role in cell adhesion to P388D1 and B16 cells and consequtive hydrophobic amino acids just aminoterminal side of the RGD sequence conforms binding site for hydroxyapatite. Analysis for heparin and fibronactin binding regions is now underway. 2)Roles of OPN in various diseases : Serum and local OPN levels were upregulated after lipopolysaccharide (LPS) stimulation and in Arthus reaction. Effect of LPS was abolished in M14M transgenic mice which show CD14 in hepatic cells under the control of metallothionein promoter and competitively u … More ptake LPS.OPN expression was detected in granuloma in lungs in transgenic mice which express TNF-alpha in lung type II epithelia under the control of surfactant promoter. Expression of OPN was strongly found in macrophages in Propionibacterium acnes induced-hepatic granuloma, whereas that in M14M mice was significantly low. Expression of OPN was not detected in islands in transgenic mice which express TNF-alpha in beta-cells under the control of insulin promoter. OPN was strongly expressed in macrophages in bovine type II collagen-induced arthritis in mice. Maternal or fetal injection of mAb resulted in abortion, suggesting inhibition of organ development. OPN was strlongly expressed in tumor cells and surrowunding macrophages. The level corresponded to metastatic and infiltrating activitis. 3)Analysis of OPN transgenic mice : Transgenic mice expressing OPN under the control of alpha antitrypsin 1 promoter showed high levels of serum OPN.Analysis of antibody production, cellmediated immunity, tumorigenesis, proliferation and metastasis are underway. Less

1)抗小鼠骨桥蛋白（OPN）单克隆抗体（mAb）的产生和分析：产生了显示不同组织反应性特征的mAb。mAb A与肾和骨细胞反应，而mAb B与肾反应，但不与骨细胞反应。使用这些mAb的分析表明，不同的反应性归因于表位的掩蔽。利用单克隆抗体和重组OPN分析功能结构域，发现RGD序列在细胞与P388 D1和B16细胞的粘附中起作用，并且因此RGD序列氨基端侧的疏水氨基酸符合羟基磷灰石的结合位点。肝素和纤连蛋白结合区的分析正在进行中。2)OPN在各种疾病中的作用：脂多糖（LPS）刺激后和Arthus反应中，血清和局部OPN水平上调。在M14 M转基因小鼠中，LPS的作用被消除，该转基因小鼠在金属硫蛋白启动子的控制下显示肝细胞中的CD 14，并且竞争性地使用 ...更多信息 在肺II型上皮细胞中表达TNF-α的转基因小鼠中，在肺肉芽肿中检测到OPN表达，所述转基因小鼠在表面活性剂启动子的控制下。在痤疮丙酸杆菌诱导的肝肉芽肿中，OPN在巨噬细胞中强烈表达，而在M14 M小鼠中，OPN的表达显著降低。在胰岛素启动子控制下在β细胞中表达TNF-α的转基因小鼠中，在岛中未检测到OPN的表达。在牛II型胶原诱导的小鼠关节炎中，OPN在巨噬细胞中强烈表达。母体或胎儿注射mAb导致流产，表明器官发育受到抑制。OPN在肿瘤细胞及周围巨噬细胞中呈强阳性表达。其水平与转移和浸润活动相对应。3)OPN转基因小鼠的分析：在α-抗胰蛋白酶1启动子控制下表达OPN的转基因小鼠显示出高水平的血清OPN。正在进行抗体产生、细胞介导的免疫、肿瘤发生、增殖和转移的分析。少

项目成果

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Yasunori Higuchi 等人：“骨桥蛋白”现代化学特刊 29. 218-224 (1996)

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