ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

CD45在吞噬细胞运动和信号转导中的作用 利亚娜·哈瓦思 白细胞共同抗原(CD45)家族是一组高分子的 含胞质蛋白的重单跨膜糖蛋白 ALL细胞质膜上表达的酪氨酸磷酸酶结构域 白细胞。有关CD45功能的最新信息表明 针对某些CD45亚型的抗体试剂具有很强的免疫抑制作用 活性，并可能有助于免疫抑制作用 治疗药物，如注射的抗胸腺细胞球蛋白 移植受者预防移植物抗宿主病 和移植排斥反应。我们之前已经证明了某些细胞外的 CD45的结构域表位参与人中性粒细胞的趋化作用。我们 利用遗传方法进行了这些观察并进行了诱变 小鼠巨噬细胞系获得低或低表达的变异克隆 检测不到CD45表达。我们发现细胞系的变种缺乏 CD45在趋化反应中的表达存在缺陷 然而，重组人C5a细胞表现出正常的随机分布 能动性。这些观察结果提示CD45可能参与了 定向(趋化)迁移是一种基本的宿主防御 对炎性刺激的反应。阐明CD45在人类免疫系统中的作用 吞噬细胞趋化作用，我们正试图分离CD45基因敲除细胞 排队。在CD45基因敲除中，回复将是微不足道的，其功能是 通过野生型和突变型CD45的转染可以评价CD45的表达 CDNA.年内，构建了CD45靶向载体，该载体 利用异源启动子驱动的新霉素基因插入 插入CD45基因第12外显子。CD45外显子12的转基因实验 靶向载体显示随机整合到基因组中的可能性很大 超出了目标集成，并表示可能需要 筛选数千个克隆，然后才能成功地重组 找到了。当其他基因出现这种情况时，更有利的是 以随机积分比为目标，用一个 无启动子靶向载体，利用基因启动子 靶基因。在外显子12基因敲除的细胞系中，蛋白质合成应该 在跨膜区之前终止并导致无功能 蛋白质(基因)。构建无启动子CD45的研究正在进行中 目标向量，研究随机集成的目标 比率，并分离CD45基因敲除。一株CD45缺陷基因的分离 细胞系将为建立有功能的 该分子在吞噬细胞趋化和宿主防御中的作用。

Role of CD45 in Phagocyte Motility and Signal Transduction Liana Harvath The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout cell line. In a CD45 knockout, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, a CD45 targeting vector was constructed that utilized insertion of a neomycin gene driven by a heterologous promoter into exon 12 of CD45. Transfection experiments with the exon 12 CD45 targeting vector showed that random integration into the genome greatly exceeded targeted integration and indicated that it may be necessary to screen several thousand clones before a successful recombinant would be found. When this situation has arisen with other genes, a more favorable targeting to random integration ratio has been obtained with a promoterless targeting vector that utilizes the genomic promoter of the target gene. In an exon 12 knockout cell line, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Studies are underway to construct a promoterless CD45 targeting vector, to investigate the targeting to random integration ratio, and to isolate a CD45 knockout. The isolation of a CD45 deficient cell line will provide a valuable tool for establishing the functional role of the molecule in phagocyte chemotaxis and host defense.