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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 廖泰慶(Tai-Ching Liao) | |
dc.contributor.author | Yee-Shuan Ho | en |
dc.contributor.author | 何宜宣 | zh_TW |
dc.date.accessioned | 2021-06-17T07:04:40Z | - |
dc.date.available | 2019-07-31 | |
dc.date.copyright | 2019-07-31 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-07-26 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72729 | - |
dc.description.abstract | 黑色素瘤是犬常見的腫瘤,最常發生於口腔,也常見於趾間、皮膚及眼睛,而犬口腔黑色素瘤與人類黏膜黑色素瘤有著許多共同的特徵。人類黏膜黑色素瘤相關之癌化基因已經有不少文獻發表,但在犬隻黑色素瘤卻沒有太多研究;現有的資料顯示,在犬黑色素瘤組織或細胞中,曾發現NRAS (Q61K和Q61R) 基因和KIT (L578P) 基因的點突變,這些基因突變也是人類黑色素瘤常見的突變熱點。本研究將探討犬黑色素瘤之基因突變並評估其相關訊息傳遞變異,同時建立這些基因突變的快速分子檢測技術,以應用於黑色素瘤組織的檢測。首先,我們對14株犬黏膜型黑色素瘤細胞進行KIT、NRAS和BRAF基因的基因突變檢測,分別在KMeC和PU細胞株中發現導致KIT L578P突變的T1736C突變,另外發現造成NRAS Q61K突變的C181A突變 (UCDK9M5及C1細胞株)及Q61R突變的A182G突變(LMeC和Wood細胞株)。其次,我們建立了穩定且快速的高解析度解離分析(HRM; High Resolution Melting),可以成功應用在細胞株、經福馬林固定石蠟包埋 (FFPE; formalin fixed paraffin embedded) 或新鮮的腫瘤組織上,篩檢基因突變。再來,我們分析這些犬黑色素瘤細胞內,KIT下游三個主要信息分子AKT、MAPK和STAT3的活性,結果發現帶有KIT L578P突變的細胞株,其PI3K/AKT路徑活性比其它細胞株高,而分別帶有兩個NRAS基因突變 (Q61K和Q61R) 的4個細胞株,它們的MAPK /ERK路徑活性較其它細胞株高。在血清飢餓 (Serum starvation) 後,UCDK9M1、UCDK9M5、LMeC和KMeC這四株犬黑色素瘤細胞仍存在不同強度AKT、MAPK和STAT3的活性;而在給予KIT的配體幹細胞生長因子(SCF; stem cell factor)刺激後,所有細胞的ERK活性都會明顯增加,但STAT3活性僅有些微的上升,另外UCDK9M5、LMeC和KMeC細胞的AKT活性則沒有明顯變化,而UCDK9M1在刺激後,AKT及STAT3的活性有最高的增加比率。總結以上的結果,在我們的犬黏膜型黑素瘤細胞中發現了幾種NRAS和KIT基因點突變,它們可能促使AKT,ERK和/或STAT3信號傳導的過度活化,而這四株細胞內的KIT訊息路徑可被SCF激活。此外,我們也建立HRM檢測技術,並初步應用於新鮮的及福馬林固定過的犬黑色素瘤組織,成功的篩檢出特定的基因突變。 | zh_TW |
dc.description.abstract | Melanoma is a malignant neoplasm, and it remains mostly an untreatable fatal disease despite developing new therapeutic modalities. Melanoma in dogs is most frequently found in the oral cavity, but the digits are other common location for these neoplasms. Canine mucosal melanoma (CMM) shares many characteristics with human mucosal melanoma (HMM). Genetic alterations have been fully described in HMM, but not in CMM. Although activating mutations in KIT, and NRAS are uncommon in CMM, but the majority of CMM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Several researches and our previous studies have shown that analogous NRAS and KIT mutations in human do occur in CMM. In this study, we investigated the gene mutations and evaluated associated signal alterations in canine melanoma. First, mutation analysis of specific genes KIT, NRAS and BRAF were done on fourteen canine melanoma (CM) cell lines by sequencing. A T1736C mutation causing L578P change on KIT were detected in KMeC and PU cells, while C181A and A182G mutations causing NRAS Q61K and Q61R change, respectively, was found in UCDK9M5, C1, LMeC and Wood cells. Next, the HRM (High resolution melting) assays were established and successfully applied to determine KIT and NRAS gene mutations in CM cells, fresh CM tissues and formalin fixed paraffin embedded (FFPE) CM tissues. Second, since AKT, MAPK and STAT3 were primary downstream signals of KIT, the activity baselines of these signals were investigated in CM cell lines. The results showed cell bearing KIT mutation (L578P) has higher activity of AKT than wild type cells. Cells bearing NRAS mutations (Q61K and Q61R) present higher ERK activities than wild type cells. In serum starved condition, the activities of AKT, MAPK and STAT3 of these four cells still variously presented. In the presence of SCF (stem cell factor), the activities of MAPK markedly enhanced and the activities of STAT3 mildly increased in four cells. Besides, the activities of AKT in UCDK9M5, LMeC and KMeC cells didn’t respond to SCF. Noticeably, SCF stimulation markedly increased the AKT, MAPK and STAT3 activities of UCDK9M1 cell. In conclusion, several NRAS and KIT gene mutations were found in CM cells and might cause AKT, ERK and/or STAT3 signaling overactivity. KIT signaling of four CM cells seem activated in response to SCF. Moreover, HRM assays were established and applied to identify these KIT and NRAS gene mutations in CM cells and CM tissues. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:04:40Z (GMT). No. of bitstreams: 1 ntu-108-R06629020-1.pdf: 3390137 bytes, checksum: 1eefebb977a5b75f04d3fd887d2e1a08 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | Chapter 1. Background and Literatures Review 1
1.1 Canine melanoma 1 1.1.1 History 1 1.1.2 Epidemiology and etiology 2 1.1.3 Cytological Characteristics with phenotypic and functional differentiation 4 1.2 Human melanoma 8 1.2.1 History 8 1.2.2 Epidemiology and Etiology 9 1.2.3 Cytological Characteristics with phenotypic and functional differentiation 10 1.3 Therapy for canine melanoma 15 1.4 High resolution melting dissolution curve analysis 17 1.4.1 Amplifying graphics 18 1.4.2 Melting dissolution curve 19 1.4.3 Standardized data 19 Chapter 2. Introduction 20 Chapter 3. Materials and methods 22 3.1 Melanoma samples collection and preparation 22 3.2 DNA extraction 23 3.3 RNA extraction and reverse transcription-polymerase chain reaction 23 3.4 Mutation detection 24 3.4.1 DNA sequencing 24 3.4.2 Restriction enzyme digestion 25 3.5 Development of HRM Analysis for KIT and NRAS gene mutations in canine melanomas 25 3.5.1 TA cloning of KIT and NRAS gene fragments 25 3.5.2 Preparing LB broth, LB agar plates with ampicillin 25 3.5.3 PCR amplification and gel extraction 26 3.5.4 Ligation, transformation and spreading plate 26 3.5.5 Blue-white screening, colony PCR and plasmid extraction 27 3.5.6 Construction of KIT and NRAS gene mutations for HRM Analysis 28 3.6 Western blot for cell signaling analysis 29 3.6.1 Preparing protein samples for evaluation of signaling base lines in canine melanoma cells 29 3.6.2 Preparing protein samples for assessment of signal alterations after SCF stimulation in canine melanoma cells 30 3.6.3 Western blot 31 Chapter 4. Results 33 4.1 Collection of canine melanoma samples 33 4.2 Detection of genetic mutations in canine melanoma 33 4.2.1 Identification of KIT and NRAS mutations in canine melanoma cell lines and tissues 33 4.2.2 Detection of KIT T1736C mutation using restriction fragment length polymorphism (RFLP) 34 4.3 Development of HRM Analysis for screening KIT and NRAS gene mutations in canine melanomas 35 4.3.2 Establishment of HRM standard curve for KIT and NRAS genes 36 4.3.3 HRM analysis for screening KIT and NRAS gene mutation in canine melanoma cell lines 38 4.3.4 HRM analysis for screening KIT and NRAS gene mutation in fresh tissues and FFPE tissues of canine melanoma 39 4.4.1 Baselines of AKT, ERK and STAT3 expression were detected in melanoma cell lines 40 Chapter 5. Discussion 43 5.1 KIT, NRAS and BRAF genetic mutations in canine melanomas 43 5.2 HRM analysis for gene mutations in canine melanomas 45 5.3 Exploring signaling alterations in canine melanomas 48 5.4 Conclusion 52 Tables 53 Table 1. 53 Table 2. 54 Table 3. 55 Table 4. 56 Figures 57 Figure 1. PCR amplification for KIT and NRAS fragments. 57 Figure 2. Map and sequence reference points of TA cloning vector. 58 Figure 3. Colony PCR for screening target inserts. 59 Figure 4. Colony PCR for confirming target inserts. 60 Figure 5. KIT L578P mutations in PU and KMeC melanoma cells. 61 Figure 6. KIT silent mutations in UCDK9M5 and HERO melanoma cells. 62 Figure 7. NRAS Q61R mutations in C1 and LMeC melanoma cells. 63 Figure 8. NRAS Q61K mutations in UCDK9M5 and WOOD melanoma cells. 64 Figure 9. NRAS Q61R and Q61K mutations in FFPE tissues. 65 Figure 10. Using RFLP to detect KIT T1736C mutation in melanoma cells. 66 Figure 11. Plasmids of KIT T1736C (L578P), KIT C1743T (silent), NRAS A182G (Q61R) and NRAS C181A (Q61K) mutations were constructed. 67 Figure 12. HRM standard curve for KIT gene and NRAS gene were established. 68 Figure 13. The HRM analysis of KIT and NRAS gene mutations in canine melanoma cells. 69 Figure 14. KIT and NRAS gene mutations of fresh canine melanoma tissues were determined by HRM analysis. 70 Figure 15. NRAS gene mutations of FFPE canine melanoma tissues were determined by HRM analysis. 71 Figure 16. The activity baselines of AKT in canine melanoma cell lines. 72 Figure 17. The activity baselines of MAPK in canine melanoma cell lines. 73 Figure 18. The activity baselines of STAT3 in canine melanoma cell lines. 74 Figure 19. The alterations of AKT activities were assessed after SCF stimulation in canine melanoma cell lines 75 Figure 20. The alterations of MAPK activities were assessed after SCF stimulation in canine melanoma cell lines 76 Figure 21. The alterations of STAT3 activities were assessed after SCF stimulation in canine melanoma cell lines 77 Figure 22. Evaluation of KIT expression in canine melanoma cell lines by RT-PCR and IP-Western. 78 References 79 | |
dc.language.iso | en | |
dc.title | 探討犬隻惡性黑色素瘤中之基因突變及相關訊息傳遞變異 | zh_TW |
dc.title | Exploring gene mutations and associated signaling alterations in canine malignant melanoma | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李繼忠(Jih-Jong Lee),林辰栖(Chen-Si Lin),王尚麟,黃威翔 | |
dc.subject.keyword | 犬黑色素瘤,基因突變,KIT,NRAS,訊息路徑, | zh_TW |
dc.subject.keyword | Canine melanoma,Gene mutations,KIT,NRAS,Signaling pathway, | en |
dc.relation.page | 85 | |
dc.identifier.doi | 10.6342/NTU201902034 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-07-29 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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