Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14466
標題: 犬NGAL抗體之製備及其於犬乳腺腫瘤表現之研究
Production of anti-neutrophil gelatinase-associated lipocalin (NGAL) antibodies and investigation of NGAL expression in canine mammary gland tumors
作者: 李芳儀
Lee, Fang-Yi
關鍵字: 
NGAL
乳腺腫瘤
mammary gland tumors
出版社: 獸醫學系暨研究所
引用: 參考文獻 1.林怡杉。犬NGAL抗體製備及腎衰竭犬之NGAL濃度與臨床進展的關聯性。國立中興大學獸醫研究所臨床組碩士論文。臺中,中華民國,2011。 2.胡譽嚴。以NGAL蛋白濃度評估犬隻急性腎損傷。國立中興大學獸醫研究所臨床組碩士論文。臺中,中華民國,2011。 3.Aghel A, Shrestha K, Mullens W, Borowski A, Tang WH. Serum neutrophil gelatinase-associated lipocalin (NGAL) in predicting worsening renal function in acute decompensated heart failure. J Card Fail 16: 49-54, 2010. 4.Barresi. Neutrophil gelatinase-associated lipocalin immunoexpression in renal tumors: Correlation with histotype and histological grade. Oncol Rep 24: 2010. 5.Barresi V, Tuccari G, Barresi G. NGAL immunohistochemical expression in brain primary and metastatic tumors. Clin Neuropathol 29: 317, 2010. 6.Bauer M, Eickhoff JC, Gould MN, Mundhenke C, Maass N, Friedl A. Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer. Breast Cancer Res Treat 108: 389-397, 2008. 7.Benazzi C, Sarli G, Galeotti M, Marcato P. Basement membrane components in mammary tumours of the dog and cat. J Comp Pathol 109: 241-252, 1993. 8.Boenisch T. Antibodies. In: Boenisch T, ed. Handbook of immunochemical staining methods. 3 rd ed. Dako Corporation., California, USA, 5-11, 2001. 9.Bolignano D, Donato V, Lacquaniti A, Fazio MR, Bono C, Coppolino G, Buemi M. Neutrophil gelatinase-associated lipocalin (NGAL) in human neoplasias: a new protein enters the scene. Cancer Lett 288: 10-16, 2010. 10.Borkham‐Kamphorst E, Drews F, Weiskirchen R. Induction of lipocalin‐2 expression in acute and chronic experimental liver injury moderated by pro‐inflammatory cytokines interleukin‐1β through nuclear factor‐κB activation. Liver International 2011. 11.Cai L, Rubin J, Han W, Venge P, Xu S. The origin of multiple molecular forms in urine of HNL/NGAL. Clinical journal of the American Society of Nephrology : CJASN 5: 2229-2235, 2010. 12.Carroll JK, Herrick B, Gipson T, Lee SP. Acute pancreatitis: diagnosis, prognosis, and treatment. Am Fam Physician 75: 1513-1520, 2007. 13.Chakraborty S, Kaur S, Muddana V, Sharma N, Wittel UA, Papachristou GI, Whitcomb D, Brand RE,Batra SK. Elevated serum neutrophil gelatinase-associated lipocalin is an early predictor of severity and outcome in acute pancreatitis. Am J Gastroenterol 105: 2050-2059, 2010. 14.Cho H, Kim JH. Lipocalin2 expressions correlate significantly with tumor differentiation in epithelial ovarian cancer. J Histochem Cytochem 57: 513-521, 2009. 15.Cleary SP, Gryfe R, Guindi M, Greig P, Smith L, Mackenzie R, Strasberg S, Hanna S, Taylor B, Langer B. Prognostic factors in resected pancreatic adenocarcinoma: analysis of actual 5-year survivors. J Am Coll Surg 198: 722-731, 2004. 16.de Las Mulas JM, Millan Y, Dios R. A prospective analysis of immunohistochemically determined estrogen receptor α and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog. Vet Pathol 42: 200-212, 2005. 17.Devireddy LR, Gazin C, Zhu X, Green MR. A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell 123: 1293-1305, 2005. 18.Fernandez CA, Yan L, Louis G, Yang J, Kutok JL, Moses MA. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients. Clin Cancer Res 11: 5390-5395, 2005. 19.Flower DR. The lipocalin protein family: structure and function. Biochem J 318: 1-14, 1996. 20.Friedl A, Stoesz S, Buckley P, Gould M. Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression. Histochem J 31: 433-441, 1999. 21.Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10: 1033, 2002. 22.Grutzner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O''Brien PC, Jones RC, Ferguson-Smith MA, Marshall Graves JA. In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature 432: 913-917, 2004. 23.Gwira JA, Wei F, Ishibe S, Ueland JM, Barasch J, Cantley LG. Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro. J Biol Chem 280: 7875-7882, 2005. 24.Han H, Bearss DJ, Browne LW, Calaluce R, Nagle RB, Von Hoff DD. Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 62: 2890-2896, 2002. 25.Hanai J, Mammoto T, Seth P, Mori K, Karumanchi SA, Barasch J, Sukhatme VP. Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells. J Biol Chem 280: 13641-13647, 2005. 26.Hraba-Renevey S, Turler H, Kress M, Salomon C, Weil R. SV40-induced expression of mouse gene 24p3 involves a post-transcriptional mechanism. Oncogene 4: 601-608, 1989. 27.Hvidberg V, Jacobsen C, Strong RK, Cowland JB, Moestrup SK, Borregaard N. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake. FEBS Lett 579: 773-777, 2005. 28.Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497, 1975. 29.Kehrer JP. Lipocalin-2: pro- or anti-apoptotic? Cell Biol Toxicol 26: 83-89, 2010. 30.Kjeldsen L, Johnsen AH, Sengelov H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 268: 10425-10432, 1993. 31.Kubben FJ, Sier CF, Hawinkels LJ, Tschesche H, van Duijn W, Zuidwijk K, van der Reijden JJ, Hanemaaijer R, Griffioen G, Lamers CB, Verspaget HW. Clinical evidence for a protective role of lipocalin-2 against MMP-9 autodegradation and the impact for gastric cancer. Eur J Cancer 43: 1869-1876, 2007. 32.Kurpisz M, Gupta SK, Fulgham DL, Alexander NJ. Production of large amounts of mouse polyclonal antisera. J Immunol Methods 115: 195-198, 1988. 33.Kuwabara T, Mori K, Mukoyama M, Kasahara M, Yokoi H, Saito Y, Yoshioka T, Ogawa Y, Imamaki H, Kusakabe T, Ebihara K, Omata M, Satoh N, Sugawara A, Barasch J, Nakao K. Urinary neutrophil gelatinase-associated lipocalin levels reflect damage to glomeruli, proximal tubules, and distal nephrons. Kidney Int 75: 285-294, 2009. 34.Lee HJ, Lee EK, Lee KJ, Hong SW, Yoon Y, Kim JS. Ectopic expression of neutrophil gelatinase‐associated lipocalin suppresses the invasion and liver metastasis of colon cancer cells. Int J Cancer 118: 2490-2497, 2006. 35.Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. The Lancet 363: 1049-1057, 2004. 36.Lim R, Ahmed N, Borregaard N, Riley C, Wafai R, Thompson EW, Quinn MA, Rice GE. Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer: NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition. Int J Cancer 120: 2426-2434, 2007. 37.Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46: 258-268, 2005. 38.Lumsden KR, Nelson AM, Dispenza MC, Gilliland KL, Cong Z, Zaenglein AL, Thiboutot DM. Isotretinoin increases skin-surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne. Br J Dermatol 165: 302-310, 2011. 39.Merlo D, Rossi L, Pellegrino C, Ceppi M, Cardellino U, Capurro C, Ratto A, Sambucco P, Sestito V, Tanara G. Cancer incidence in pet dogs: findings of the Animal Tumor Registry of Genoa, Italy. J Vet Intern Med 22: 976-984, 2008. 40.Mira E, Lacalle RA, Buesa JM, de Buitrago GG, Jimenez-Baranda S, Gomez-Mouton C, Martinez-A C, Manes S. Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface. J Cell Sci 117: 1847-1857, 2004. 41.Misdorp W. Tumors of the mammary gland. In: Meuten D, ed. Tumors in domestic animals. 4th ed. A Blackwell Publishing Company., Iowa, USA, 575-606, 2002. 42.Mishra J. Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Early Urinary Biomarker for Ischemic Renal Injury. J Am Soc Nephrol 14: 2534-2543, 2003. 43.Mishra J, Dent C, Tarabishi R, Mitsnefes M, Ma Q, Kelly C, Ruff S, Zahedi K, Shao M, Bean J. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. The Lancet 365: 1231-1238, 2005. 44.Moestrup SK, Verroust PJ. Megalin-and cubilin-mediated endocytosis of protein-bound vitamins, lipids, and hormones in polarized epithelia. Annu Rev Nutr 21: 407-428, 2001. 45.Moniaux N, Chakraborty S, Yalniz M, Gonzalez J, Shostrom VK, Standop J, Lele SM, Ouellette M, Pour PM, Sasson AR, Brand RE, Hollingsworth MA, Jain M, Batra SK. Early diagnosis of pancreatic cancer: neutrophil gelatinase-associated lipocalin as a marker of pancreatic intraepithelial neoplasia. Br J Cancer 98: 1540-1547, 2008. 46.Mori K. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest 115: 610-621, 2005. 47.Nelson AM, Zhao W, Gilliland KL, Zaenglein AL, Liu W, Thiboutot DM. Neutrophil gelatinase–associated lipocalin mediates 13-cis retinoic acid–induced apoptosis of human sebaceous gland cells. J Clin Invest 118: 1468-1478, 2008. 48.Overley B, Shofer FS, Goldschmidt MH, Sherer D, Sorenmo KU. Association between ovarihysterectomy and feline mammary carcinoma. J Vet Intern Med 19: 560-563, 2005. 49.Ramalho L, Ribeiro-Silva A, Cassali G, Zucoloto S. The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms. Vet Pathol 43: 424-429, 2006. 50.Rodvold JJ, Mahadevan NR, Zanetti M. Lipocalin 2 in cancer: when good immunity goes bad. Cancer Lett 316: 132-138, 2012. 51.Schmidt-Ott KM, Mori K, Kalandadze A, Li JY, Paragas N, Nicholas T, Devarajan P, Barasch J. Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia. Curr Opin Nephrol Hypertens 15: 442-449, 2006. 52.Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 18: 407-413, 2007. 53.Schneider R. Comparison of age, sex, and incidence rates in human and canine breast cancer. Cancer 26: 419-426, 1970. 54.Smith ER, Zurakowski D, Saad A, Scott RM, Moses MA. Urinary biomarkers predict brain tumor presence and response to therapy. Clin Cancer Res 14: 2378-2386, 2008. 55.Soni SS, Cruz D, Bobek I, Chionh CY, Nalesso F, Lentini P, de Cal M, Corradi V, Virzi G, Ronco C. NGAL: a biomarker of acute kidney injury and other systemic conditions. Int Urol Nephrol 42: 141-150, 2010. 56.Sorenmo KU, Shofer FS, Goldschmidt MH. Effect of spaying and timing of spaying on survival of dogs with mammary carcinoma. J Vet Intern Med 14: 266-270, 2000. 57.Stoesz SP, Friedl A, Haag JD, Lindstrom MJ, Clark GM, Gould MN. Heterogeneous expression of the lipocalin NGAL in primary breast cancers. Int J Cancer 79: 565-572, 1998. 58.Venkatesha S, Hanai Ji, Seth P, Karumanchi SA, Sukhatme VP. Lipocalin 2 Antagonizes the Proangiogenic Action of Ras in Transformed Cells. Mol Cancer Res 4: 821-829, 2006. 59.Wang Y, Lam KS, Kraegen EW, Sweeney G, Zhang J, Tso AW, Chow WS, Wat NM, Xu JY, Hoo RL, Xu A. Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans. Clin Chem 53: 34-41, 2007. 60.Yan L, Borregaard N, Kjeldsen L, Moses MA. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem 276: 37258-37265, 2001. 61.Yan QW, Yang Q, Mody N, Graham TE, Hsu CH, Xu Z, Houstis NE, Kahn BB, Rosen ED. The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance. Diabetes 56: 2533-2540, 2007. 62.Yang J, Goetz D, Li JY, Wang W, Mori K, Setlik D, Du T, Erdjument-Bromage H, Tempst P, Strong R. An iron delivery pathway mediated by a lipocalin. Mol Cell 10: 1045-1056, 2002. 63.Yang J, Bielenberg DR, Rodig SJ, Doiron R, Clifton MC, Kung AL, Strong RK, Zurakowski D, Moses MA. Lipocalin 2 promotes breast cancer progression. Proc Natl Acad Sci USA 106: 3913-3918, 2009. 64.Yasukawa T, Kanei-Ishii C, Maekawa T, Fujimoto J, Yamamoto T, Ishii S. Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. J Biol Chem 270: 25328-25331, 1995. 65.Zerega B, Cermelli S, Michelis B, Cancedda R, Descalzi Cancedda F. Expression of NRL/NGAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation. Eur J Cell Biol 79: 165-172, 2000.
摘要: Neutrophil gelatinase-associated lipocalin(NGAL)為一分子量約25 kDa的醣蛋白,其主要在調控細胞增生、分化及凋亡等機制中扮演了重要的角色。近年來的許多研究皆指出NGAL藉由誘導上皮細胞間質化轉型路徑(EMT),並藉由競爭鐵離子使腫瘤細胞存活率上升等途徑,進而促進了腫瘤形成以及遠端轉移,這些結果開啟了研究NGAL是否可作為乳腺腫瘤的惡性程度及預後的評估指標。而本研究之目的為製備犬NGAL單源抗體,運用在犬乳腺腫瘤之免疫組織化學染色,並期望能運用至其他犬臨床樣本檢測。 首先利用pET32b表現系統製備出之犬重組NGAL蛋白,用以免疫實驗小鼠,將免疫後小鼠脾臟細胞取出與NS-1小鼠骨髓瘤細胞融合,形成可產生抗體且能持續繼代之融合瘤細胞。以ELISA及西方轉漬法篩選出具有強陽性的融合瘤細胞株進行擴養、以限制稀釋的方式將其單株化、並以此細胞株接種小鼠誘導產生富含單源抗體之腹水。實驗結果顯示,此單源抗體可成功的以西方轉漬法辨識到腎衰竭犬及乳腺腫瘤犬尿液中之NGAL,此外應用於免疫組織化學染色法(IHC) ,可成功的偵測到犬乳腺腫瘤中的NGAL。由於此單源抗體的效力與本實驗室既有之兔子來源多株抗體效力相當,確定兩者皆可用於免疫組織化學染色。 用自製的犬NGAL抗體,以IHC分析法偵測並評估所蒐集的209個犬正常乳腺(77個)及乳腺腫瘤組織(132個)之NGAL表現量;其評分方式根據陽性反應細胞量以及染色強度評分。染色結果可見NGAL陽性反應主要表現於腫瘤細胞中,將其結果評分後進行分析統計,NGAL表現量在正常乳腺、良性及惡性腫瘤中有顯著的差異(p < 0.005);其中惡性腫瘤有最高的NGAL表現。此外,在carcinoma(屬惡性腫瘤)中NGAL的表現量顯著高於adenoma (屬良性腫瘤)。另外本研究中也發現,雖然整體而言惡性腫瘤組織有較高的NGAL,但是患惡性腫瘤的狗,其身上的正常乳腺之NGAL表現量卻與良性腫瘤犬的正常乳腺相當,並無全面性提升NGAL 量的傾向,而比較來自兩種犬隻的良性腫瘤也有相同結果。綜合上述結果推測,NGAL具有發展成為一個評估乳腺腫瘤指標的潛力。
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.
URI: http://hdl.handle.net/11455/14466
其他識別: U0005-0808201212395500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0808201212395500
Appears in Collections:獸醫學系所

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
TAIR Related Article
 
Citations:


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.