权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Studies on mechanisms of auto-immune disease in dogs

犬自身免疫性疾病机制研究

基本信息

批准号：
05454126
负责人：
YAMADA Takatsugu
金额：
$ 2.75万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Monoclonal antibodies for canine T cell surface antigen are important for research of canine auto-immune disease. In this study, we tried making monoclonal antibody for canine T cell, because of a few monoclonal antibodies were available for the research.Spleen cells from immunized mouse with canine thymo-cytes were fused with murine myeloma cells (P3X63-Ag.653) by polyethelenglycol method. Eleven strains of hybridomas that producing monoclonal antibody against canine lymphocytes were established. Three antibodies produced by these hybridomas were characterized by immuno-precipitation of cell surface antigen, functional analysis of each antibody positive cells and distribution of positive cells in tissues and blood. Monoclonal antibody (MAb) 7F1,6H6 and 8C12 were the IgG1 (k), IgG3 (k) and IgG1 (k) isotype of mouse immunoglobulin respectively. MAb 7F1 and 6h6 precipitated a 72 kDa, 55 kDa monomeric protein respectively from the surface of peripheral blood lymphocytes (PBMC) under reduc … More ing conditions. MAb 8C12 precipitated 33 kDa and 38kDa hetero dimer protein from the surface of PBMC.7F1 positive cells were rich in medulla of thymus, on the other hand 6H6 or 8C12 positive cells were rich in cortex of thymus respectively by immunohistochemistry. In functional assay, proliferative responses was enhanced in enrichment of 7F1 or 6H6 positive cells by sorting with cell-sorter. 8C12 positive cells were lower proliferative response than 8C12 negative cells. IL-2 activities by biological assay using CTLL-2 cells were higher in 7F1 or 6H6 positive cells, however the activities were lower in 8C12 positive cells compared to each antibody negative cells. MAb 7F1,6H6 and 8C12 recognized 63.7(]SY.+-。[)2.9,42.5(]SY.+-。[)3.6 and 14.5(]SY.+-。[)1.0% of PBMC respectively by cell analyzer.From these results, we concluded that 7F1,6H6 and 8C12 recognized CD5, CD4 and CD8 on canine PBMC respectively. Characterization of the other antibodies (derived from 8 hybridomas) are continuing now.These three antibodies are useful for analysis of cell surface antigen, imunohistochemistry and functional studies of canine lymphocytes. We are using these antibodies for studies of inducing oral tolerance, peripheral blood analysis of autoimmune-disease dogs and immunohistochemistry of intestinal and tracheobronchial mucosa. Less

犬T细胞表面抗原单克隆抗体对犬自身免疫性疾病的研究具有重要意义。由于目前可供研究的单克隆抗体很少，本研究尝试制备犬T细胞单克隆抗体。用聚乙二醇法将犬胸腺细胞免疫小鼠脾细胞与小鼠骨髓瘤细胞（P3X63-Ag.653）融合。建立了11株产生抗犬淋巴细胞单克隆抗体的杂交瘤。通过细胞表面抗原的免疫沉淀、各抗体阳性细胞的功能分析和阳性细胞在组织和血液中的分布，对这些杂交瘤产生的三种抗体进行了表征。单克隆抗体（MAb） 7F1、6H6和8C12分别为小鼠免疫球蛋白的IgG1 (k)、IgG3 (k)和IgG1 (k)同型。MAb 7F1和MAb 6h6在还原条件下分别从外周血淋巴细胞（PBMC）表面析出72kda和55kda的单体蛋白。MAb 8C12从pbmc表面析出33 kDa和38kDa的异二聚体蛋白，7f1阳性细胞富集于胸腺髓质，6H6和8C12阳性细胞分别富集于胸腺皮层。在功能实验中，通过细胞分选器对7F1或6H6阳性细胞进行富集，增强了增殖反应。8C12阳性细胞的增殖反应低于8C12阴性细胞。用CTLL-2细胞进行生物测定，IL-2活性在7F1和6H6阳性细胞中较高，而在8C12阳性细胞中活性低于抗体阴性细胞。MAb 7F1、6H6和8C12分别通过细胞分析仪识别63.7(]SY.+- .[)2.9、42.5(]SY.+- .[)3.6和14.5(]SY.+- .[)1.0%的PBMC。结果表明，7F1、6H6和8C12分别在犬PBMC上识别CD5、CD4和CD8。其他抗体（来自8个杂交瘤）的鉴定正在继续。这三种抗体可用于犬淋巴细胞的细胞表面抗原分析、免疫组织化学和功能研究。我们将这些抗体用于诱导口服耐受性的研究，自身免疫性疾病犬的外周血分析以及肠道和气管支气管粘膜的免疫组织化学研究。少