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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李雅珍(Ya-Jane Lee) | |
dc.contributor.author | Po-Han Wu | en |
dc.contributor.author | 吳柏翰 | zh_TW |
dc.date.accessioned | 2021-06-17T03:43:56Z | - |
dc.date.available | 2018-02-23 | |
dc.date.copyright | 2018-02-23 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-02-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70108 | - |
dc.description.abstract | Neutrophil gelatinase-associated lipocalin (NGAL)是一種具發展性的腎病標誌物,在人和狗的研究中發現,NGAL 以 monomer, dimer 和 NGAL/MMP-9 複合體的形式存在於不同的泌尿道疾病。人類醫學研究已經探討 Matrix metalloproteinases-9 (MMP-9) 在慢性腎臟病 (chronic kidney disease, CKD) 中,關於腎臟纖維化過程所扮演的角色。但是貓尿中不同NGAL分子形態和泌尿道疾病之相關性,以及 MMP-9 在貓隻腎臟疾病所扮演的角色還尚未清楚,而成為本研究的目的。
從2014年9月到2017年5月,總共有159隻貓納入實驗,並且分成以下不同組別:(1)健康貓、(2)慢性腎臟病貓、(3)急性腎損傷貓、(4)有氮血症和膿尿的貓、(5)膿尿的貓(6)沒有任何腎臟泌尿道疾病但有其他疾病的貓。以non-reducing Western blotting 來分析尿中不同的 NGAL 分子型態。尿中 NGAL 和 MMP-9 的濃度則以實驗室建立的 sandwich ELISA 來測量。並且計算尿中NGAL 和 MMP-9 濃度和尿中肌酸酐 (creatinine) 的比值 (uNGAL-to-creatinine ratio, UNCR; uMMP-9-to-creatinine ratio, UMCR)。 根據Western blotting的結果,貓尿中可以偵測到不同分子型態的NGAL,同時觀察到MMP-9的存在。NGAL monomer和MMP-9 monomer的出現會和血中濃度上升的BUN 和creatinine有關。NGAL monomer在急性腎損傷(acute kidney injury, AKI)的貓,出現頻率會比CKD的貓還高。IRIS CKD分級比較高(stage 3 and 4)的貓,尿中出現的NGAL monomer比例會比健康貓和IRIS CKD分級較低的貓還要高。尿中NGAL dimer的出現會和尿中的白血球數目有關。而NGAL dimer的出現則是和尿中白血球數目、膿尿和泌尿道感染(urinary tract infection, UTI)有顯著相關。此研究中,大部分的貓(126/159, 79.2%)甚至是健康貓(7/12, 58.3 %),在尿中有NGAL/MMP-9 complex的存在。 以實驗室自製的sandwich ELISA確認尿中的uNGAL, uMMP-9, UNCR和UMCR數值後,結果顯示氮血症但是沒有膿尿的貓,會比健康貓有更高的UNCR, uMMP-9和UMCR (下述以median [IQR]表示, UNCR: 0.87 [1.98] x10-6 vs. 0.17 [0.24] x10-6; uMMP-9: 2.51 [10.59] ng/mL vs. 0.11 [1.23] ng/mL; UMCR: 3.91 [12.95] x10-6 vs. 0.03 [0.78] x10-6)。 AKI的貓會比CKD的貓有較高的uNGAL和UNCR。以receiver operating characteristic (ROC) 分析發現uNGAL和UNCR可以被用來區分AKI和CKD,利用uNGAL和UNCR來區分AKI和CKD時的ROC曲線下面積(AUROC)分別是0.877和0.825。IRIS CKD分級越高的貓,有越高的uNGAL濃度和UNCR數值。而膿尿的貓也會比健康貓有較高的uNGAL, uMMP-9, UNCR和UMCR數值。 根據研究結果,uNGAL和uMMP-9在貓都可以作為泌尿道疾病的生物標識,然而,同時也應該要考慮不同的uNGAL和uMMP-9分子形態,來了解是否源自不同的泌尿道異常。貓尿中的NGAL monomer和MMP-9 monomer可能和腎臟損傷有關,而貓尿中出現NGAL dimer則可能和膿尿有關。 | zh_TW |
dc.description.abstract | Neutrophil gelatinase-associated lipocalin (NGAL), a promising renal biomarker, exists as monomer, dimer and/or NGAL/MMP-9 complex forms with different urinary diseases in humans and dogs. The role of matrix metalloproteinases-9 (MMP-9) in renal fibrosis of chronic kidney disease (CKD) has been investigated in human medicine. Both the different molecular forms of urine NGAL in feline with different urinary diseases and the role of MMP-9 in naturally occurring feline renal diseases were never investigated and would be the purpose of the present study.
From September 2014 to May 2017, 159 cats were enrolled and classified into different groups as follows: (1) healthy cats, (2) cats with chronic kidney disease (CKD), (3) cats with acute kidney injury (AKI), (4) cats with azotemia and pyuria, (5) cats with pyuria (6) cats without any urinary diseases but with other diseases. Western blotting with non-reducing condition was performed to analyze the different molecular forms of NGAL in urine samples. The concentrations of uNGAL and uMMP-9 were also measured by our in-house sandwich ELISA with the record of uNGAL-to-creatinine ratio (UNCR) and uMMP-9-to-creatinine ratio (UMCR). According to the results of Western blotting, three different NGAL molecular forms and the MMP-9 monomer were present in cat urine. The appearance of NGAL monomer and MMP-9 monomer were both associated with the elevated levels of serum BUN and creatinine. Moreover, the presence frequency of urine NGAL monomer and MMP-9 monomer are both higher in cats with azotemia than cats without azotemia. Cats with AKI had higher presence frequency of NGAL monomer than cats with CKD did. The proportion of cats with urinary NGAL monomer was higher in CKD IRIS stage 3 and stage 4 than in the control group and CKD IRIS stage 2. The presence of dimeric NGAL was associated with the urine WBC number, Pyuria, and urinary tract infection (UTI). Most cats (126/159, 79.2%) and even more than half of the healthy cats (7/12, 58.3 %) in the present study had NGAL/MMP-9 complex in urine. After determining the values of uNGAL, uMMP-9, UNCR and UMCR by the in-house sandwich ELISA, the results showed that the UNCR, uMMP-9 and UMCR of cats with AKI or CKD were higher than those of healthy cats (The following data are median [IQR], UNCR: 0.87 [1.98] x10-6 vs. 0.17 [0.24] x10-6; uMMP-9: 2.51 [10.59] ng/mL vs. 0.11 [1.23] ng/mL; UMCR: 3.91 [12.95] x10-6 vs. 0.03 [0.78] x10-6). Cats with AKI had higher levels of uNGAL and UNCR than cats with CKD did (The following data are median [IQR], uNGAL: 1.78 [5.81] ng/mL vs. 0.37 [0.37] ng/mL; UNCR: 2.92 [6.56] x10-6 vs. 0.56 [1.15] x10-6). The receiver operating characteristic (ROC) analysis revealed that the uNGAL and UNCR can be used to differentiate AKI from CKD. The area under the ROC curves of uNGAL and UNCR for differentiating AKD from CKD are 0.877 and 0.825, respectively. The levels of uNGAL, UNCR and UMCR in cats at CKD IRIS stage 3 and stage 4 were higher than those in cats at CKD IRIS stage 2 and healthy cats. The uNGAL, uMMP-9, UNCR and UMCR of cats with pyuria were higher than those of the healthy cats. In conclusion, both uNGAL and uMMP-9 appear to be urinary biomarkers in cats, however, different molecular forms of uNGAL and uMMP-9 should be simultaneously considered to understand the origin of different urinary abnormality. The urinary NGAL monomer and MMP-9 monomer seem to be associated with the kidney injury in cats, whereas the presence of NGAL dimer in urine appears to be related to pyuria in cats. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:43:56Z (GMT). No. of bitstreams: 1 ntu-107-R03643012-1.pdf: 4145854 bytes, checksum: 49c750e410c660ad7cab8bf454227c82 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii ABSTRACT v CONTENT viii LIST OF FIGURES xiii LIST OF TABLES xiv Chapter 1 Introduction 1 Chapter 2 Literature review 4 2.1 Introduction of NGAL 4 2.1.1 Nature and Cellular Sources of NGAL in Humans 4 2.1.2 Expression of NGAL in multiple tissues 4 2.2 Functional roles of NGAL 5 2.2.1 NGAL in infection 5 2.2.2 NGAL in inflammation 6 2.2.3 NGAL in immunity 7 2.2.4 NGAL in cell differentiation 7 2.2.5 NGAL in metabolism 8 2.2.6 NGAL in bone homeostasis 8 2.3 Clinical application of NGAL in human medicine 9 2.3.1 NGAL in Acute Kidney Injury (AKI) 9 2.3.1.1 Cardiac surgery associated with AKI (CSA-AKI) 10 2.3.1.2 Contrast-induced nephropathy (CIN) 11 2.3.1.3 AKI with sepsis patients 11 2.3.2 NGAL in Chronic kidney disease (CKD) 12 2.3.2 NGAL in urinary tract infection (UTI) 13 2.4 Introduction of MMP-9 14 2.5 Functional roles of MMP-9 14 2.6 Clinical application of MMP-9 in human medicine 15 2.7 Three different molecular forms of NGAL 17 2.7.1 NGAL monomer 17 2.7.2 NGAL dimer 18 2.7.3 Heterodimer (NGAL conjugated with MMP-9) 19 2.8 Clinical application of NGAL in veterinary medicine 21 2.8.1 Urine NGAL in dogs with AKI 21 2.8.2 NGAL for prognostic prediction in dogs with CKD 22 2.8.3 NGAL in dogs with lower urinary tract diseases (LUTD) 23 2.8.4 NGAL in dogs with neoplasia 23 2.8.5 Different forms of urine NGAL in dogs with urinary disease 24 2.8.6 NGAL in cats with CKD 25 2.9 MMP-9 in veterinary medicine 26 Chapter 3 Materials and Methods 27 3.1 Patients and Sample Collection 27 3.1.1 Case grouping criteria- based on the clinical parameters 27 3.1.2 Case grouping criteria- based on the present molecular form 30 3.1.3 Sample collection, preparation and storage 31 3.2 Establishment of sandwich ELISA for detecting feline NGAL and MMP-9 in urine samples 32 3.2.1 Preparation of antibody against feline NGAL 32 3.2.2 Preparation of antibody against feline MMP-9 32 3.2.2.1 Confirmation of the cross-reaction between canine and feline MMP-9 32 3.2.2.2 Purification and quantitation of recombinant canine MMP-9 protein 33 3.2.2.3 Protocol of recombinant MMP-9 purification 34 3.2.3 Protocol of sandwich ELISA for detecting feline NGAL or MMP-9 35 3.2.4 Evaluation of the precision of in-house ELISA 38 3.3 Western blotting for detecting feline NGAL & MMP-9 38 3.3.1 Sample preparation 38 3.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) 38 3.3.3 Western blotting 39 3.4 Statistical analysis 41 Chapter 4 Results 42 4.1 Patients and Sample Collection 42 4.2 Establishing of ELISA for detecting feline MMP-9 43 4.2.1 Quantitative analysis for purified recombinant canine MMP-9 protein 43 4.2.2 Production of antibodies against MMP-9 46 4.3 Determine the concentration of feline NGAL and MMP-9 in urine samples by sandwich ELISA 47 4.3.1 Establishment of the standard curves for NGAL and MMP-9 49 4.3.2 The precision of sandwich ELISA 49 4.4 Detection of feline NGAL and MMP-9 in urine by Western blotting 50 4.4.1 Confirmation of different molecular forms of NGAL 50 4.4.2 Confirmation of presence of NGAL/MMP-9 complex and MMP-9 monomer 50 4.5 Results of statistical analysis 53 4.5.1 Difference of the urinary NGAL and MMP-9 molecular forms among groups 53 4.5.1.1 Comparisons among groups associated with azotemia 53 4.5.1.2 Comparisons among groups associated with non-renal related diseases 55 4.5.1.3 Comparisons among AKI, CKD, pyuria and Other groups 57 4.5.2 The concentration of Urinary NGAL and MMP-9 59 4.5.2.1 Urinary NGAL concentration (uNGAL) 59 4.5.2.2 Urinary NGAL-to-creatinine ratio (UNCR) 59 4.5.2.3 Urinary MMP-9 concentration (uMMP-9) 60 4.5.2.4 Urinary MMP-9-to-creatinine ratio (UMCR) 60 4.5.3 Receiver operating curve (ROC) analyses 64 4.5.4 Comparison between Azo and control group 66 4.5.5 Comparison between cats with and without azotemia 70 4.5.6 Comparison between cats with and without pyuria 72 4.5.7 Parameters between the groups with and without different molecular form of NGAL and MMP-9 present in urine 74 4.5.7.1 NGAL monomer 74 4.5.7.2 NGAL dimer 76 4.5.7.3 NGAL/MMP-9 complex 78 4.5.7.4 MMP-9 monomer 80 4.5.8 Comparison among cats with CKD stage 2-4 82 4.5.9 Progression of CKD 84 4.5.10 Dimeric NGAL and urinary tract infection (UTI) 86 Chapter 5 Discussion 88 5.1 Urinary NGAL and MMP-9 in feline renal disease 89 5.2 Urinary NGAL and MMP-9 between cats with AKI and CKD 90 5.3 Urinary NGAL and MMP-9 among cats with CKD IRIS stage 2-4 92 5.4 Urinary NGAL and MMP-9 in feline CKD progression 94 5.5 Urinary NGAL and MMP-9 in cats with pyuria 94 5.6 Urinary dimeric NGAL and urinary MMP-9 in cats with UTI 97 5.7 Urinary NGAL and MMP-9 in cats with non-urinary diseases 98 Chapter 6 Conclusion 100 Reference 101 | |
dc.language.iso | en | |
dc.title | 貓尿中NGAL和MMP-9於不同泌尿道疾病之分子形態和濃度 | zh_TW |
dc.title | Different molecular forms and concentration of urine neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinases-9 (MMP-9) in cats with different urinary diseases | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐維莉(Wei-Li Hsu),吳允升(Vin-Cent Wu),蔡沛學(Jason Tsai) | |
dc.subject.keyword | 分子型態,生物標記,NGAL,MMP-9,貓泌尿道疾病, | zh_TW |
dc.subject.keyword | molecular forms,Biomarker,NGAL,MMP-9,feline urinary disease, | en |
dc.relation.page | 115 | |
dc.identifier.doi | 10.6342/NTU201800092 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-02-04 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 臨床動物醫學研究所 | zh_TW |
顯示於系所單位: | 臨床動物醫學研究所 |
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