Molecular action mechanism of eosinophil granule proteins in destruction of mucosal cells in ulcerative colitis

嗜酸细胞颗粒蛋白破坏溃疡性结肠炎粘膜细胞的分子作用机制

• 批准号: 12670500
• 负责人: MAKIYAMA Kazuya
• 金额: $ 2.18万
• 依托单位: Nagasaki University Hospital
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670500/
• 关键词: ulcerative colitis; eosinophil cationic protein: ECP; polypeptides; polyclonal antibody; eosinophil cationic protein

To investigate the role of activated eosinophils in the inflammatory process of ulcerative colitis, we prepared antibodies against polypeptides of a human eosinophil granule protein, eosinophil cationic protein (ECP), in attempt to purify ECP.1. The following four ECP polypeptides were selected based on the human ECP amino acid sequence reported by Barker et al.: ECP1, CRYADRPGRRFVVA; ECP2, CDNRDPRDSPRYPVV; ECP3, CTIAMRAINNYRWRC; and ECP4, TRAQWFAIQHISLNPPR.2. Rabbits were immunized with ECP polypeptide antigens, and anti-human ECP antibodies that reacted with synthetic peptides even the rabbit antisera were diluted to 1/10,000 were obtained. 3. None of the acquired anti-human ECP antibodies reacted as anti-ECP antiserum in an ECP assay system using Pharmacia ECP RIA kit. 4. Anti-human ECP2 antibody and anti-human ECP3 antibody reacted with eosinophils and monocytes in immunohistochemical staining.The above findings suggested that although it is necessary to confirm whether the acquired anti-ECP antibodies react with human ECP, at least the ECP2 and ECP3 amino acid sequence regions have immunological reactivity. The anti-inflammatory effect of the anti-human ECP antibodies should be investigated using an experimental animal model.

为了研究活化的嗜酸性粒细胞在溃疡性结肠炎炎症过程中的作用，我们制备了针对人嗜酸性粒细胞颗粒蛋白、嗜酸性粒细胞阳离子蛋白(ECP)的多肽的抗体，试图纯化ECP.1。根据Barker等人报道的人ECP氨基酸序列，筛选出以下4种ECP多肽：ECP1、CRYADRPGRRFVVA； ECP2、CDNRDPRDSPRYPVV； ECP3、CTIARAINNYRWRC；和 ECP4、TRAQWFAIQHISLNPPR.2。用ECP多肽抗原免疫兔子，获得即使兔抗血清稀释至1/10,000也能与合成肽反应的抗人ECP抗体。 3.获得的抗人ECP抗体均未在使用Pharmacia ECP RIA试剂盒的ECP测定系统中作为抗ECP抗血清反应。 4.免疫组化染色中抗人ECP2抗体和抗人ECP3抗体与嗜酸性粒细胞和单核细胞发生反应。上述结果表明，虽然需要确认获得的抗ECP抗体是否与人ECP反应，但至少ECP2和ECP3氨基酸序列区域具有免疫反应性。应使用实验动物模型研究抗人 ECP 抗体的抗炎作用。