In vivo analysis of osteopontin function

1)抗小鼠骨桥蛋白（OPN）单克隆抗体（mAb）的制备与分析：制备出具有不同组织反应性的单克隆抗体。单抗A与肾细胞和骨细胞反应，而单抗B与肾细胞反应，但不与骨细胞反应。利用这些单抗分析表明，不同的反应性归因于表位的掩蔽。利用单抗和重组OPN对功能域进行分析，发现RGD序列在细胞粘附P388D1和B16细胞中发挥作用，相应的疏水氨基酸恰好在RGD序列的氨基端符合羟基磷灰石的结合位点。肝素和纤动蛋白结合区分析正在进行中。2) OPN在各种疾病中的作用：血清和局部OPN水平在脂多糖（LPS）刺激和Arthus反应后上调。M14M转基因小鼠在金属硫蛋白启动子的控制下，肝细胞中显示CD14， LPS的作用被消除。在表面活性剂启动子控制下，表达tnf - α的转基因小鼠肺II型上皮肉芽肿中检测到了OPN的表达。在痤疮丙酸杆菌诱导的肝肉芽肿中，巨噬细胞中强烈表达OPN，而在M14M小鼠中则明显低表达。在胰岛素启动子控制的β细胞中表达tnf - α的转基因小鼠中，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，巨噬细胞强烈表达OPN。母体或胎儿注射单克隆抗体导致流产，提示器官发育受到抑制。OPN在肿瘤细胞和周围巨噬细胞中强烈表达。该水平对应于转移性和浸润性活动。3) OPN转基因小鼠分析：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。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