| Summary: | 碩士 === 國立中興大學 === 獸醫學系暨研究所 === 100 === Neutrophil gelatinase-associated lipocalin (NGAL), a 25-kDa glycoprotein, plays the role in cell multiplication, differentiation and apoptosis. Recent studies indicated that NGAL involves in tumorigenesis and metastasis of breast cancer by inducing epithelial mesenchymal transition (EMT) in cells and promotes cancer cells survival by scavenging iron. These results revealed NGAL may serve as a biomarker of breast cancer prognosis. Aims of this study were to generate monoclonal antibodies for detection of canine NGAL with immunohistochemistry (IHC). Initially, recombinant thioredoxin-NGAL fusion protein expressed in pET32b vector was used as antigen to immunize mice. The spleen cells from immunized mice were fused with NS-1 myeloma cell line to generate hybridoma cells producing antibodies. The hybridoma cells positively producing NGAL specific antibody as screened by analysis of ELISA and western blotting were then cloned by limiting dilutions. To obtain high volume of monoclonal antibodies, the cloned hybridoma cells were intraperitoneal injected in mice to produce ascites. By western blotting, the monoclonal antibody successfully detected the expression of NGAL in urine of dogs with renal failure and tumor cells of mammary gland tumor (MGT), respectively. Furthermore, in IHC assay, the staining pattern of this monoclonal antibody was similar to that stained with the rabbit polyclonal NGAL antibodies manufactured in our laboratory. Detection of NGAL in 209 canine specimens, including normal mammary gland tissues (77) and mammary gland tumors (132) by IHC revealed that NGAL mainly expressed in cytoplasm of mammary gland epithelial tumor cells. Classification score of NGAL staining data was according to the percentage of tumor cell with positive staining and stain intensity. In statistical analysis, NGAL expression was significantly different between normal mammary glands and tumors, including malignant tumors and benign tumors (p < 0.005); the highest expression level was found in malignant tumors. In addition, NGAL expression in carcinoma (malignant) was also higher than that in adenoma (benign). Moreover, in dogs with malignant MGT, the NGAL level in their normal counterparts was similar to that of dog with benign MGT, suggesting that the overexpression of NGAL expression is limited in malignant tumors region. Collectively, in this study, monoclonal NGAL antibody was generated, and NGAL may be a useful marker in the evaluation of canine mammary gland tumors.
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