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Functional analysis of α-fetoprotein by genetically engineered mice

基因工程小鼠甲胎蛋白的功能分析

基本信息

批准号：
12670127
负责人：
NISHI Shinzo
金额：
$ 2.18万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

α-fetoprotein (AFP) is a serum protein in appreciable amounts in fetal but not in adult serum. However, serum AFP is frequently elevated in patients with hepatocellular carcinomas and yolk sac tumors. Numerous studies have suggested that immunomodulation may be one of the biological functions of AFP. To clarify the biological function of the AFP in vivo, we have established two transgenic mice, which have human and mouse AFP genes under the control of β-actin promoter. In these mice, ubiquitous expressions of AFP were observed. Using these mice, we have been analyzed experimental autoimmune diseases, such as arthritis, autoimmune thyroiditis and experimental allergic encephalomyelitis. We have showed that development of all these experimental autoimmune diseases were significantly suppressed in AFP produced transgenic mice compared with wild mice. The development of experimental allergic encephalomyelitis of the mouse AFP producing mice was more suppressed than the mouse producing human AFP.Monoclonal antibodies (MAbs) against human AFP have been used in a variety of procedures including immuno assay, immunoscintigraphy and immunotherapy. A number of MAbs have been produced, but their epitopes remain to be defined. We have analyzed 36 MAbs against human AFP using recombinant AFP peptides and synthesized overlapping octapeptides. Epitopes of 13 MAbs were mapped in domain I and 17 were on domain II. The epitope of one of the MAb, AFY6, was localized on C^<175>KAENAVE^<182>.We also analyzed the AFP gene regulation. Glucocorticoid inhibit rodent AFP gene activity but stimulate expression of the human AFP. The glucocorticoid induction of human AFP is mediated by a GRE on the AFP gene promoter and corresponding element of the mouse is a binding site of the HNF4, a hepatocyte enriched transcription factor. The interaction of the glucocorticoid receptor and HNF4 may account for this opposite effects.

甲胎蛋白（AFP）是一种血清蛋白，在胎儿血清中含量可观，但在成人血清中含量不高。然而，血清甲胎蛋白经常升高，肝细胞癌和卵黄囊肿瘤患者。大量研究表明，免疫调节可能是AFP的生物学功能之一。为了阐明AFP在体内的生物学功能，我们建立了两个转基因小鼠，分别将人和小鼠AFP基因置于β-actin启动子的控制下。在这些小鼠中，观察到AFP的普遍表达。使用这些小鼠，我们已经分析了实验性自身免疫性疾病，如关节炎，自身免疫性甲状腺炎和实验性变态反应性脑脊髓炎。我们已经表明，与野生小鼠相比，所有这些实验性自身免疫性疾病的发展在AFP产生的转基因小鼠中被显着抑制。实验性变态反应性脑脊髓炎的发生在产生人AFP的小鼠身上，而在产生人AFP的小鼠身上，其发生受到更大的抑制。已经产生了许多单克隆抗体，但它们的表位仍有待确定。我们分析了36个单克隆抗体抗人AFP重组AFP肽和合成重叠八肽。13个单克隆抗体的表位定位在结构域I，17个定位在结构域II。其中一株单抗AFY 6的表位定位于C^<175>KAENAVE^<182>上，并对AFP基因的调控进行了分析。糖皮质激素抑制啮齿类动物AFP基因的活性，但刺激人AFP的表达。人AFP的糖皮质激素诱导由AFP基因启动子上的GRE介导，小鼠的相应元件是肝细胞富集的转录因子HNF 4的结合位点。糖皮质激素受体和HNF 4的相互作用可能解释了这种相反的作用。