In vivo analysis of osteopontin function

1)制备并分析了具有不同组织反应性的抗鼠骨桥蛋白(OPN)单抗。单抗A与肾脏和骨细胞反应，而单抗B与肾脏反应，但不与骨细胞反应。对这些单抗的分析表明，不同的反应性可归因于表位的掩蔽。利用mAb和重组OPN对功能域进行分析，发现RGD序列在细胞与P388D1和B16细胞的黏附中起作用，由此产生的疏水氨基酸仅在RGD序列的氨基末端与羟基磷灰石结合。目前正在对肝素和纤维粘连蛋白结合区进行分析。2)骨桥蛋白在多种疾病中的作用：脂多糖(LPS)刺激后，血清和局部骨桥蛋白水平升高，参与阿尔萨斯反应。在金属硫蛋白启动子和竞争性u…控制下表达CD14的M14M转基因小鼠中，内毒素的作用被取消在表面活性启动子控制下的肺II型上皮细胞表达肿瘤坏死因子-α的转基因小鼠肺肉芽肿中可检测到更多pTake LPS.OPN表达。在痤疮丙酸杆菌诱导的肝肉芽肿小鼠的巨噬细胞中，骨桥蛋白的表达较强，而在M14M小鼠的巨噬细胞中表达较低。在胰岛素启动子控制下的β细胞中表达肿瘤坏死因子-α的转基因小鼠未检测到骨桥蛋白的表达。在小鼠II型胶原诱导的关节炎模型中，OPN在巨噬细胞中呈强阳性表达。母体或胎儿注射单抗会导致流产，表明器官发育受到抑制。OPN在肿瘤细胞和周围巨噬细胞中呈强阳性表达。该水平与转移和浸润性活动性相对应。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的转基因小鼠血清OPN水平较高，抗体产生、细胞免疫、肿瘤发生、增殖和转移的分析正在进行中。较少

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