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

• 批准号: 3770439
• 负责人: L HARVATH
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3770439
• 关键词: CD antigens; biological signal transduction; cell motility; chemotaxis; clone cells; complementary DNA; flow cytometry; gene mutation; myelocyte; phagocytes; phosphoprotein phosphatase; recombinant proteins; transfection

The CD45 antigen family is a group of high molecular weight glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. We have previously shown that epitopes of CD45 are involved in neutrophil chemotaxis to the chemoattractants, leukotriene B4 and recombinant human C5a (rHuC5a). The purpose of this project is to generate and characterize stable CD45 negative myeloid cell clones and determine the role of CD45 in phagocyte function. The Abelson-murine leukemia virus transformed macrophage cell line, RAW 264, was selected for this project because RAW 264 macrophages exhibit dose-dependent chemotaxis to rHuC5a. RAW 264 cells were mutagenized with the alkylating agent, ethyl methanesulfonate, for 24 hours in tissue culture flasks. Mutagenized cells were scraped off the flask, replated into several flasks, and allowed to grow in Minimal Essential Medium containing 10% fetal calf serum. Cells were harvested from the flasks when they reached confluent growth and were treated with a biotinylated-monoclonal antibody (mAb) to murine CD45. Streptavidin- conjugated magnetic beads were incubated with the CD45 mAb-treated cells and the mixture was run over a magnetized column to enrich for the CD45 negative cells. After multiple rounds of CD45 negative selection and expansion of the enriched population in culture, 75 CD45 negative clones were identified by flow cytometric analysis. Twenty clones were screened for chemotactic responsiveness to rHuC5a and >90% of the clones had defective chemotactic responses. Current studies are in progress to transfect the CD45 negative mutants with CD45 cDNA and determine whether normal chemotactic function returns upon expression of CD45. These results indicate that CD45 expression on macrophage plasma membranes is important for normal chemotactic responsiveness to rHuC5a. The CD45 negative RAW 264 mutant clones provide a unique cellular system to probe the functional activity of CD45 in myeloid cells.

CD 45抗原家族是一组高分子量糖蛋白 在所有白细胞的质膜上表达。 CD 45具有 蛋白酪氨酸磷酸酶活性，并似乎调节信号 通过特异性结合转导和淋巴细胞活化 T和B淋巴细胞上的受体分子。 我们之前已经证明， CD 45的表位参与中性粒细胞趋化性， 化学引诱剂、白三烯B4和重组人C5 a（rHuC 5a）。 的 本项目的目的是产生和表征稳定的CD 45 阴性髓系细胞克隆，并确定CD 45在吞噬细胞中的作用 功能 Abelson-小鼠白血病病毒转化的巨噬细胞 本项目选择RAW 264细胞系，因为RAW 264巨噬细胞 对rHuC 5a表现出剂量依赖性趋化性。 RAW 264细胞 用烷化剂甲磺酸乙酯诱变24小时 在组织培养瓶中。 将诱变的细胞从烧瓶上刮下， 重新接种到几个烧瓶中，并使其在最小必需培养基中生长。 含有10%胎牛血清的培养基。 从细胞中收获细胞。 当它们达到汇合生长时，用 抗鼠CD 45的生物素化单克隆抗体（mAb）。 链霉亲和素- 将缀合的磁珠与CD 45 mAb处理的细胞一起孵育 并将混合物在磁化柱上运行以富集CD 45 阴性细胞。 在多轮CD 45阴性选择和 扩增培养物中的富集群体，75个CD 45阴性克隆 通过流式细胞术分析鉴定。 筛选了20个克隆 对于rHuC 5a的趋化反应性，>90%的克隆具有 趋化反应缺陷。 目前正在进行研究， 用CD 45 cDNA检测CD 45阴性突变体，并确定是否 在表达CD 45时恢复正常的趋化功能。 这些结果 表明巨噬细胞质膜上CD 45表达是重要的 对rHuC 5a的正常趋化反应。 CD 45阴性RAW 264 突变体克隆提供了一种独特的细胞系统来探测功能性 骨髓细胞中CD 45的活性。