臺灣博碩士論文加值系統

Matrix metalloprotease-9 (MMP-9)促進細胞外基質的降解，被認為與腫瘤的侵犯及轉移有關，並且可與neutrophil gelatinase-associated lipocalin (NGAL)形成複合物。本篇研究目的為調查MMP-9、NGAL及其複合物於犬乳腺腫瘤之表現。本實驗搜集自70隻犬之血清檢體及57份尿液檢體。血清及尿液檢體皆經由西方墨點法偵測MMP-9及NGAL表現，其中130 kDa蛋白質為MMP-9/NGAL複合物。結果顯示乳腺腫瘤患犬及無腫瘤犬之血清檢體的表現無明顯差異。約80%乳腺腫瘤犬（包含良性及惡性腫瘤）之尿液檢體可偵測到MMP-9/NGAL複合物，但此表現在無腫瘤犬之尿液檢體中未能偵測到。少數乳腺腫瘤患犬之尿液檢體可偵測到92 kDa 的MMP-9 單體、25 kDa 的NGAL單體、46 kDa的NGAL雙體及70 kDa的NGAL三聚體。經本實驗結果得到MMP-9、 NGAL及其複合物在血清及尿液中的表現與乳腺腫瘤類別、分期、分級及其他臨床特徵無明顯相關。然而，在尿液中MMP-9/NGAL 複合物表現與否與乳腺腫瘤組織中是否受到炎症細胞浸潤有顯著相關。臨床上顯示MMP-9/NGAL複合物不會表現於無腫瘤犬隻的尿液中，但無法作為準確預測乳腺腫瘤患犬進程及癒後的指標。

Matrix metalloprotease-9 (MMP-9), which accelerates breakdown of extracellular matrix associated with tumor invasion and metastasis, can form a high molecular weight complex with neutrophil gelatinase-associated lipocalin (NGAL). The aim of this study was to investigate the expression of MMP-9, NGAL and their complex in dogs with mammary gland tumor. In total, 70 serum and 57 urine samples from 70 dogs were included in this study. Both NGAL and MMP-9 in serum and urine were detected by western blotting. The 130 kDa molecular weight protein was assigned as MMP-9/NGAL complex. There was non-significant difference of MMP-9/NGAL complex present in serum between dogs with mammary gland tumor and tumor-free dogs. In urine samples, MMP-9/NGAL complex was detected of almost 80% of dogs with either malignant or benign mammary gland tumor, but not in urine from tumor-free dogs. The 92 kDa monomer form of MMP-9 and the 25 kDa monomer, 46 kDa dimer and 70 kDa timer form of NGAL were detected in a few urinary samples. The results of expressions of MMP-9, NGAL and their complex in urine and serum were not associated with the type of mammary gland tumor, staging, grading and other clinical characteristics, whereas significant correlation were observed between expression of urinary MMP-9/NGAL complex and mammary gland tumors with infiltration of inflammatory cells according to histopathology. Clinically, these data revealed that MMP-9/NGAL complex was not expressed in urine of tumor-free dog but detection of MMP-9, NGAL and their complex in urine may not predict the progression and prognosis of canine mammary gland tumors.

致謝 i
摘要 ii
Abstract iii
List of abbreviations vii
List of Tables viii
List of Figures ix
Chapter 1. Introduction 1
Chapter 2. Materials and methods 8
2.1. Sample collection 8
2.2. Large scale expression and purification of His-tagged recombinant NGAL and MMP-9 proteins 9
2.2.1 Preparation of crude extract 9
2.2.2 Preparation of the column for purification 10
2.2.3 Procedure of Ni-NTA chromatography 12
2.2.4 Analysis of efficiency of protein purification by SDS-PAGE 13
2.2.5 Dialysis of the purified recombinant NGAL and MMP-9 proteins 13
2.2.6 Protein quantification 14
2.3 Detection of NGAL and MMP-9 in serum and urine sample 14
2.3.1 Clinic sample preparation 14
2.3.2 SDS-PAGE 14
2.3.3 Western blotting 16
2.4 Statistical analysis 17
Chapter 3. Results 18
3.1 Animals 18
3.2 Large scale expression and purification of His-tagged recombinant NGAL and MMP-9 proteins 19
3.3 Protein quantification 21
3.4 Detection NGAL and MMP-9 expression by western blot with mouse anti-NGAL antibody and rabbit anti-MMP-9 antibody. 22
3.4.1 Expression of NGAL and MMP-9 in serum samples 22
3.4.2 Expression of NGAL and MMP-9 in urine samples 24
Chapter 4. Discussion 28
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