人類精胺酸酉每生物活性探討:單株抗體製備、免疫功能探討以及臨床免疫病檢測之研究

SUMMARY Arginase converts L-arginine into urea and L-ornithine in the urea cycle. Recent studies have indicated that arginase participates in the regulation of immuno-response. It was proved that arginase is associated with many clinical diseases. However, the pathophisiological role of arginase is not well understood. The present study was aimed to establish a specific and sensitive method to assay the arginase concentration of plasma or cell culture. We also investigated the effects of arginase activity on immuno-functions and the cytotoxicity of arginase in tumor cells. The detection of arginase concentration is also applied to assay the clinical immuno-relative diseases and malignancies. In the present study, arginase was extract from the human liver by 45-70% saturated ammonium-sulfate and purified by affinity chromatography and molecular sieve methodology. A mouse monoclonal antibody against human liver arginase (anti-arginase I mAb), was produced serving as the first antibody, and a horseradish peroxidase-conjugated polyclonal rabbit anti-arginase antibody was produced to serve as the secondary antibody to establish the sandwich enzyme-linked immunoassay (ELISA) for arginase concentration. The cytotoxicity study of arginase on tumor cells showed that arginase could inhibit the PHA induced ?PBMC proliferation and B-lymphocyte’s Ig-G secretion. The production of TNF-?? by PBMC was also inhibited and which was dose-dependent. The expresses of IL-2R, CD3, CD4 and CD8 in the cell surface were also decreased by arginase treatment, suggesting that arginase could hamper T and B lymphocytes proliferation and inhibit the phagocytosis. Study showed arginase could kill liver cancer cell line; which showed the more differential was the more efficient. The arginase efficiently inhibited K562 and Raji cells proliferations but not U937 cells. The arginase concentration in cancer cell was apparently higher than in PBMC. Using the highly sensitive and specific sandwich ELISA assay, we found that both serum arginase activity and protein levels were significantly higher in patients with rheumatoid arthritis (RA) than in patients with systemic lupus erythematosus (SLE) or osteoarthritis (OA) or in healthy controls. Arginase protein concentrations in supernatants of monocyte cultures from RA patients were also significantly higher than in those from SLE or OA patients or healthy controls. Meanwhile, the activities of intracellular arginase in monocytes from RA patients were significantly higher than in monocytes from healthy controls. In RA patients, there was a significant correlation between the serum concentrations of arginase protein and rheumatoid factor (r=0.82, pl0.0001). These data indicate that increased arginase production is seen in RA patients, but not in other immune-related diseases, suggesting that increased arginase production is unique to, and may play an important role in, the pathogenesis of RA disease. Besides, we found that the serum arginase concentration was significantly lower in patients with squamous cell carcinoma (SCC) than in healthy controls (p=0.00077). However, the serum arginase concentrations were significantly higher in patients with esophageal cancer (ECA), gastric cancer (GCA), colon cancer (colon Ca) and lymphoma than in healthy controls (pl0.05, pl0.05, pl0.05, pl0.05, respectively). These data indicate that arginase may also play an important role in the pathogenesis of these malignancies.