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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 廖泰慶(Albert Tai-ching Liao) | |
| dc.contributor.author | Lu-Ping Lu | en |
| dc.contributor.author | 呂律秉 | zh_TW |
| dc.date.accessioned | 2021-06-16T04:02:49Z | - |
| dc.date.available | 2020-02-03 | |
| dc.date.copyright | 2015-02-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-10-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55444 | - |
| dc.description.abstract | 惡性黑色素瘤為犬隻常見的口唇部腫瘤，其為一致死性疾病並有原位侵略及高度轉移的特性，現今常見的治療方式有手術切除、化學療法及放射線治療，這些治療方式預後通常不好，因此發展針對犬黑色素瘤的嶄新治療方式在現階段是十分必要的。在本研究中我們建立出一套犬黑色素瘤藥物篩選平台並運用此平台篩選小分子抑制劑Dasatinib及新興烷基化藥物。首先，針對八個犬黑色素瘤細胞株進行表現型的檢測：針對特定基因KIT、NRAS及BRAF利用PCR/DNA定序技術進行突變分析，其結果發現於UCDK9M3、KMeC及C1三株犬黑色素細胞俱有KIT T1736C的基因點突變，造成KIT L579P的氨基酸突變，於UCDK9M5犬黑色素細胞發現NRAS C181A基因點突變，造成NRAS Q61K的氨基酸突變。另外，也針對數個黑色素腫瘤的標的蛋白進行免疫細胞化學染色，S100及KIT蛋白在所有九株細胞的表現皆可被觀察到，KIT下游蛋白NRAS則在不同細胞的表現及活性各有所不同，phospho-ERK 和phospho-AKT之活性利用“cell array”染色在所有犬黑色素瘤細胞上皆無法被觀察到。接著於細胞活性實驗，投與小分子抑制劑Dasatinib及新興烷基化藥物BO-1055、BO-2094、BO-1055、BO-1978後，對於KIT L579P、NRAS Q61K及wild type犬黑色素瘤細胞的活性皆有不同程度的抑制，其中又以Dasatinib及BO-1978效果最好。經由細胞週期分析及Annexin V細胞凋亡實驗發現投與Dasatinib會造成細胞生長週期停止於G1期並使細胞進行凋亡，投與BO-1978與BO-2094則造成G2/M細胞週期的停止並使細胞進行凋亡。最後，在SCID mice接種UCDK9M5、KMeC及CM01黑色素腫瘤細胞的生物體試驗中，投與BO-1978造成UCDK9M5和CM01團塊大小顯著的消退。在本研究中，我們針對八株黑色素瘤細胞進行表現型的鑑定，並投與Dasatinib及BO-1978，發現藥物俱有篩選上的潛力，或許未來能成為應用在臨床上治療犬黑色素瘤的新發展藥物。 | zh_TW |
| dc.description.abstract | Malignant melanoma is most common oral malignancy in dogs. It is a fatal disease along with local invasiveness and frequent metastasis. The poor response has been observed with all treatments including surgical removal, chemotherapy and radiation therapy. Thus, developing novel therapeutic approach for canine malignant melanoma is necessary. In this study, we established a drug-screening platform and used that platform to evaluate small molecule inhibitor, Dasatinib, and novel alkylating agents for therapy of canine melanoma. First of all, various phenotypes of eight canine melanoma cell lines were examined in this study. Mutation analysis of specific target genes KIT, NRAS and BRAF were done by PCR/DNA sequencing and KIT T1736C mutation causing KIT L579P change was detected in UCDK9M3, KMeC and C1 cells, while NRAS C181A mutation causing NRAS Q61K change was found in UCDK9M5 cell. Moreover, several target proteins expressions were investigated. Expressions of S100 and KIT were observed in all of the canine melanoma cell lines. KIT downstream protein NRAS was identified in several cell lines with different expression levels. However, no phospho-ERK and phospho-AKT were identified in any cell lines by “cell array”. Secondly, in cell viability assay, different melanoma cells revealed different response when treated with therapeutic drugs, in which Dasatinib and Bo-1978 effectively decreased the cell viability. Thirdly, cell cycle analysis and Annexin V apoptosis assay revealed that the treatment of Dasatinib causes a G1 cell cycle arrest and induction of apoptosis and also the treatment of BO-1978 and BO-2094 caused a G2/M cell cycle arrest and induction of apoptosis. At last, in SCID mice xenograft model of UCDK9M5, KMeC and CM01 cells, the treatment of BO-1978 significantly inhibited the growth of UCDK9M5 and CM01. In this study, we identified the phenotypes of eight melanoma cells and evaluated target drug Dasatinib and novel alkylating agents. Our data suggest that Dasatinib and BO-1978 may potentially be selected as a candidate for therapy of canine melanoma. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T04:02:49Z (GMT). No. of bitstreams: 1 ntu-103-R01629020-1.pdf: 7207726 bytes, checksum: 197b0695dee30f25ba2595f2c278a74a (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 口試委員審定書 I
誌謝 II 中文摘要 III Abstract IV Chapter 1. Background and Literatures Review 1 1.1 Canine melanoma 1 1.1.1 History 1 1.1.2 Etiology 2 1.1.3 Cytological Characteristics with phenotypic and functional differentiation 4 1.2 Human melanoma 7 1.2.1 History 7 1.2.2 Etiology 8 1.2.3 Cytological Characteristics with phenotypic and functional differentiation 10 1.3 Therapy for canine melanoma 15 1.3.1 Surgery removal 15 1.3.2 Radiation therapy 15 1.3.3 Chemotherapy 16 1.3.4 Immunotherapy 17 1.3.5 DNA vaccine 18 1.3.6 Small molecule inhibitor 19 Chapter 2. Introduction 21 Chapter 3. Materials and methods 25 3.1 Melanoma samples collection and preparation 25 3.2 RNA extraction and reverse transcription-polymerase chain reaction 26 3.3 Mutation detection 27 3.3.1 DNA sequencing 27 3.3.2 Restriction enzyme digestion 27 3.4 Immunohistochemistry and Immunocytochemistry 28 3.5 Colony formation assay and cell micro-array assay 30 3.6 Small molecule inhibitor and novel alkylating agents 30 3.7 Cell viability assay 31 3.8 Cell cycle analysis 31 3.9 Annexin V apoptosis assay 32 3.10 Western blot 33 3.11 Wound healing assay 35 3.12 SCID mice xenograft model 36 3.13 Statistical analysis 36 Chapter 4. Results 37 4.1 The establishment and collection of canine melanoma cell lines 38 4.2 The genetic mutation detection of canine melanoma 38 4.2.1 Presence of KIT C1743T (silent) and T1736C (L579P) mutations and NRAS C181A (Q61K) mutation 38 4.2.2 Restriction enzyme detection of KIT T1736C mutation 39 4.3 The intracellular signaling protein expression of canine melanoma 39 4.3.1 S100 and KIT expression were detected in all melanoma cell lines while NRAS was identified with different expression level 39 4.3.2 Establishment of “cell micro-array” assay 40 4.4 The biological function of canine melanoma cells 41 4.4.1 Proliferation of melanoma cells in minimum concentration of FBS 41 4.4.2 The morphology and colony formation of melanoma cells 41 4.5 Evaluation of therapeutic drugs on rapid screening platform 42 4.5.1 Dasatinib inhibits cell viability and motility, induces cell cycle arrest and apoptosis in melanoma cells through the inhibition of ERK pathway 43 4.5.2 Novel alkylating agents BO-1978 and BO-2094 inhibits cell viability, induces cell cycle arrest and apoptosis in melanoma cells through the diminishment of PARP 44 4.6 SCID mice xenograft model 45 Chapter 5. Discussion 46 Tables 53 Figures 55 References 81 | |
| dc.language.iso | en | |
| dc.subject | 新興烷基化藥物 | zh_TW |
| dc.subject | 免疫細胞化學染色 | zh_TW |
| dc.subject | NRAS | zh_TW |
| dc.subject | KIT | zh_TW |
| dc.subject | 基因點突變 | zh_TW |
| dc.subject | Dasatinib | zh_TW |
| dc.subject | 犬黑色素瘤 | zh_TW |
| dc.subject | DNA point mutation | en |
| dc.subject | Dasatinib | en |
| dc.subject | Novel alkylating agent | en |
| dc.subject | Immunocytochemistry | en |
| dc.subject | NRAS | en |
| dc.subject | KIT | en |
| dc.subject | Canine melanoma | en |
| dc.title | 犬黑色素瘤細胞表現型鑑定及對腫瘤治療藥物的評估 | zh_TW |
| dc.title | Identification of Phenotype and Evaluation of Cancer Therapeutic Drugs in Canine Melanoma Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林辰栖(Chen-Si Lin),劉振軒(Chen-Hsuan Liu),張仕杰(Shih-Chieh Chang) | |
| dc.subject.keyword | 犬黑色素瘤,基因點突變,KIT,NRAS,免疫細胞化學染色,新興烷基化藥物,Dasatinib, | zh_TW |
| dc.subject.keyword | Canine melanoma,DNA point mutation,KIT,NRAS,Immunocytochemistry,Novel alkylating agent,Dasatinib, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-10-16 | |
| 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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