ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

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在吞噬细胞中的作用 趋化性，我们正试图分离CD45基因敲除突变细胞系。 在CD45基因敲除突变体中，回复将可以忽略不计，并且功能 通过野生型和突变型CD45的转染可以评价CD45的表达 CDNA.年内，Lambda克隆由不同的片段组成 用12号外显子寡核苷酸探针和2个 获得阳性克隆。得到了详细的限制图 每个lambda克隆并用于构建CD45基因敲除 带有外显子12的载体被新霉素基因破坏。在外显子12的基因敲除中 突变型，蛋白质合成应在跨膜区之前终止 并产生一种无功能的蛋白质(基因)。细胞已经被转基因 利用CD45基因敲除载体和PCR筛选程序 突变细胞的检测已经开发出来了。目前正在进行研究，以 从转染体中分离出CD45缺陷突变细胞。这个 CD45缺陷细胞系的分离将为 确定分子在吞噬细胞趋化中的功能作用 和东道主防守。

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 mutant cell line. In a CD45 knockout mutant, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, lambda clones comprised of various fragments of the CD45 gene were screened with an exon 12 oligonucleotide probe and two positive clones were obtained. Detailed restriction maps were derived for each lambda clone and were used in the construction of a CD45 knockout vector with exon 12 disrupted by a neomycin gene. In an exon 12 knockout mutant, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Cells have been transfected with the CD45 knockout vector and a PCR screening procedure for the detection of a mutant cell has been developed. Studies are underway to isolate the CD45 deficient mutant cell from the transfectants. 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.