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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃敏銓 | |
| dc.contributor.author | Yi-Ting Lin | en |
| dc.contributor.author | 林怡廷 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:37:39Z | - |
| dc.date.available | 2022-02-24 | |
| dc.date.copyright | 2017-02-24 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2017-02-10 | |
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Phelan, C.M., et al., Polymorphism in the GALNT1 gene and epithelial ovarian cancer in non-Hispanic white women: the Ovarian Cancer Association Consortium. Cancer Epidemiol Biomarkers Prev, 2010. 19(2): p. 600-4. 32. Li, C., et al., GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self-Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells. Cancer Res, 2016. 76(5): p. 1273-83. 33. Ramzan, M., et al., N-acetylgalactosaminyltransferases in cancer. Oncotarget, 2016. 34. Block, H., K. Ley, and A. Zarbock, Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice. J Immunol, 2012. 188(11): p. 5674-81. 35. Zhang, W. and H.T. Liu, MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res, 2002. 12(1): p. 9-18. 36. Mebratu, Y. and Y. Tesfaigzi, How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer? Cell Cycle, 2009. 8(8): p. 1168-75. 37. 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D'Arcangelo, D., et al., PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omics approach. Oncotarget, 2016. 43. Maehana, S., et al., Suppression of lymphangiogenesis by soluble vascular endothelial growth factor receptor-2 in a mouse lung cancer model. Biomed Pharmacother, 2016. 84: p. 660-665. 44. Pothula, S.P., et al., Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer. Br J Cancer, 2016. 114(3): p. 269-80. 45. Organ, S.L. and M.S. Tsao, An overview of the c-MET signaling pathway. Ther Adv Med Oncol, 2011. 3(1 Suppl): p. S7-s19. 46. Soares, H.P., et al., Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2. Mol Cancer Ther, 2015. 14(4): p. 1014-23. 47. Chakedis, J., et al., Characterization of RON protein isoforms in pancreatic cancer: implications for biology and therapeutics. Oncotarget, 2016. 7(29): p. 45959-45975. 48. Cavel, O., et al., Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor. Cancer Res, 2012. 72(22): p. 5733-43. 49. Amit, M., et al., Upregulation of RET induces perineurial invasion of pancreatic adenocarcinoma. Oncogene, 2017. 50. Sugiyama, Y., et al., Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms. Pol J Pathol, 2016. 67(2): p. 136-44. 51. Moschovis, D., G. Bamias, and I. Delladetsima, Mucins in neoplasms of pancreas, ampulla of Vater and biliary system. World J Gastrointest Oncol, 2016. 8(10): p. 725-734. 52. Osako, M., et al., Immunohistochemical study of mucin carbohydrates and core proteins in human pancreatic tumors. Cancer, 1993. 71(7): p. 2191-9. 53. Saitou, M., et al., MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas. J Clin Pathol, 2005. 58(8): p. 845-52. 54. Park, J.H., et al., Critical roles of mucin 1 glycosylation by transactivated polypeptide N-acetylgalactosaminyltransferase 6 in mammary carcinogenesis. Cancer Res, 2010. 70(7): p. 2759-69. 55. Chou, C.H., et al., Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway. Oncotarget, 2015. 6(8): p. 6123-35. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59781 | - |
| dc.description.abstract | 胰臟癌的死亡率在台灣十大癌症中排名第八。被診斷罹患胰臟癌的病人，其五年存活率只有5％。異常的醣化作用(glycosylation)會影響許多細胞的特性，包括:細胞的增生、侵入和轉移能力等。醣基轉移酶(glycosyltransferases)表現的改變，是癌症的特徵之一。N-acetylgalactosaminyltransferase 1 (GALNT1)會調節黏蛋白型氧型醣化作用(mucin-type O-glycosylation)，並促進膀胱和胰臟癌的惡性特性。公共資料庫中的數據顯示，在胰臟癌的組織中，GALNT1mRNA 表現量較正常的胰臟組織高。然而，GALNT1 在胰臟癌中所扮演的角色仍然不清楚。本研究中我們證實在HPAC胰臟癌細胞中，抑制GALNT1 表現，會抑制細胞之生長、移動及侵入能力。此外，抑制GALNT1 表現，並藉由10％胎牛血清(fetal bovine serum, FBS)或肝細胞生長因子(hepatocyte growth factor receptor, HGFR)誘導，會抑制肝細胞生長因子受體和ERK 的磷酸化。以上的實驗結果，證實抑制GALNT1 表現，會抑制胰臟癌細胞的惡性特性，而肝細胞生長因子受體，可能參與調節此過程。我們也構築pET-30a/GALNT1 質體(plasmid)，藉由大腸桿菌誘導產生GALNT1-His Tag 重組蛋白作為抗原，並透過皮下注射方式施打紐西蘭白兔，使其產生免疫反應。由西方墨點實驗，證實抗血清中含有對GALNT1-His Tag 重組蛋白產生特異性的抗體。未來，將會進一步純化GALNT1 多株抗體(polyclonal antibody)，期望可應用於GALNT1 的研究。 | zh_TW |
| dc.description.abstract | Pancreatic cancer is the eighth of the leading cause of cancer-related deaths in Taiwan.The five-year survival rate is only 5% for pancreatic cancer patients. Aberrant glycosylation can influence many cellular properties including cell proliferation,migration and invasion. Altered expression of glycosyltransferases is one of the unique characteristics of cancers. N-acetylgalactosaminyltransferase 1(GALNT1) initiates mucin-type O-glycosylation. It has been reported to promote the malignant character of bladder and liver cancer cells. Public databases showed that the expression of GALNT1 mRNA is up-regulated in pancreatic tumors compared withnormal pancreas. However, the role of GALNT1 in pancreatic cancer is still unclear. In this study, we showed that knockdown of GALNT1 inhibited cell growth, migration and invasion in HPAC pancreatic cancer cells. Furthermore, knockdown of GALNT1 inhibited phosphorylation of hepatocyte growth factor receptor (HGFR) and ERK induced by 10% fetal bovine serum (FBS) or HGF. Taken together, these findings suggest that GALNT1 knockdown inhibits the malignant character of pancreatic cancer cells, and the HGFR signaling pathway could be involved in this process. In addition, we constructed GALNT1-pET plasmid and produced GALNT1-His Tag recombinant protein by E.coli. The GALNT1-His Tag recombinant protein was used as an antigen to immunize New Zealand white rabbits. Western blot analysis validated the specificity of anti-serum against GALNT1-
His Tag recombinant protein, indicating that this GALNT1 polyclonal antibody can be used for future studies on GALNT1. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:37:39Z (GMT). No. of bitstreams: 1 ntu-105-R03446002-1.pdf: 2786231 bytes, checksum: 4681cd1aae615559103573a5458aaf66 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書....................................................................................................... I
致謝 .................................................................................................................................II 中文摘要 ........................................................................................................................ III Abstract.......................................................................................................................... IV 目 錄 ..............................................................................................................................V 圖目錄 ...........................................................................................................................VII 表目錄 ..........................................................................................................................VIII 一、前言.......................................................................................................................... 1 1.1 胰臟癌 .................................................................................................................. 1 1.2 蛋白質的醣化作用(Protein glycosylation) .......................................................... 2 1.3 黏蛋白型醣化作用(Mucin-type glycosylation) .................................................. 2 1.4 蛋白質的醣化作用(Protein glycosylation)和癌症 .............................................. 3 1.5 N-acetylgalactosamine (GalNAc)-transferases (GALNTs)和癌症 ...................... 3 1.6 N-acetylgalactosamine (GalNAc)-transferases (GALNTs)和胰臟癌 .................. 5 1.7 N-acetylgalactosamine(GalNAc)-transferases 1 (GALNT1)的介紹 ................... 5 1.8 Mitogen-activated protein kinase (MAPK)訊號路徑調控細胞的增生能力 ...... 6 1.9 Mitogen-activated protein kinase (MAPK)訊號路徑和癌症 .............................. 6 1.10 受體酪胺酸激酶(Receptor tyrosine kinase) ....................................................... 7 1.11 受體酪胺酸激酶(Receptor tyrosine kinase)跟癌症 ........................................... 8 1.12 受體酪胺酸激酶(Receptor tyrosine kinase)跟胰臟癌 ....................................... 8 二、研究目標.................................................................................................................. 9 三、材料和方法............................................................................................................ 10 3.1 細胞株和細胞培養 ............................................................................................ 10 3.2 siRNA 抑制GALNT1 表現 .............................................................................. 10 3.3 mRNA 萃取 ........................................................................................................ 10 3.4 反轉錄聚合連鎖反應(Reverse Transcription Polymerase Chain Reaction, RTPCR) 合成cDNA ............................................................................................. 11 3.5 即時定量聚合酶連鎖反應(Real-time Polymerase Chain Reaction, Real-time- PCR) ................................................................................................................. 11 3.6 細胞蛋白質的萃取及定量 ................................................................................ 12 3.7 西方墨點法(Western blot analysis) .................................................................. 12 3.8 人類受體酪胺酸激酶磷酸化實驗(Human phospho-receptor tyrosine kinase Array) ............................................................................................................... 13 3.9 錐蟲藍染色法(Trypan blue exclusion assay) .................................................. 14 3.10 細胞移動能力分析(Transwell migration assay) ............................................. 14 3.11 細胞侵入能力分析(Matrigel invasion assay) ................................................. 14 3.12 構築pET-30a /GALNT1 質體 ......................................................................... 15 3.13 生產GALNT1-His Tag 重組蛋白 .................................................................... 15 3.14 純化GALNT1-His Tag 重組蛋白 .................................................................... 16 3.15GALNT1 多株抗體(polyclonal antibody)的製造 ............................................. 16 四、結果........................................................................................................................ 17 4.1 在HPAC 胰臟癌細胞中，20 個GALNTs 基因家族的mRNA 表現量。 ..... 17 4.2 公共資料庫顯示在正常胰臟和胰臟癌組織中GALNT1 的mRNA 表現量。 ......................................................................................................................... 17 4.3 GALNT1 siRNA 抑制HPAC 胰臟癌細胞GALNT1 的表現。 ...................... 18 4.4 抑制GALNT1 表現會抑制HPAC 胰臟癌細胞生長能力。 .......................... 18 4.5 抑制GALNT1 表現會抑制HPAC 胰臟癌細胞其移動(Migration)和侵入 (Invasion)能力。 ............................................................................................. 18 4.6 抑制GALNT1 表現會抑制HPAC 胰臟癌細胞中AKT 和ERK 的磷酸化。 ......................................................................................................................... 19 4.7 抑制GALNT1 表現對HPAC 胰臟癌細胞中人類受體酪胺酸激酶磷酸化的影 響。 ................................................................................................................. 19 4.8 抑制GALNT1 表現會抑制HPAC 胰臟癌細胞中HGFR 的表現量。 .......... 20 4.9 構築pET-30a/GALNT1 質體。 ........................................................................ 21 4.10 誘導和製造GALNT1-His Tag 重組蛋白。 .................................................... 21 4.11GALNT1 血清對於GALNT1-His Tag 重組蛋白具有特異性。 .................... 21 五、討論........................................................................................................................ 22 六、參考文獻................................................................................................................ 26 圖一、在 HPAC 胰臟癌細胞中，20 個GALNT 家族基因的mRNA 表現量。 . 30 圖二、公共資料庫顯示在正常胰臟和胰臟癌組織中GALNT1 的mRNA 表現 量。 ................................................................................................................. 31 圖三、GALNT1 siRNA 抑制HPAC 胰臟癌細胞GALNT1 的表現。 ................. 32 圖四、抑制GALNT1 表現對HPAC 胰臟癌細胞生長能力的影響。 .................. 33 圖五、抑制GALNT1 表現對HPAC 胰臟癌細胞其移動(Migration)和侵入 (Invasion)能力的影響。 ................................................................................. 34 圖六、抑制GALNT1 表現對HPAC 胰臟癌細胞中AKT、ERK 的磷酸化影 響。 ................................................................................................................. 35 圖七、抑制GALNT1 表現對HPAC 胰臟癌細胞中人類受體酪胺酸激酶磷酸化 的影響。 ......................................................................................................... 36 圖八、抑制GALNT1 表現對HPAC 胰臟癌細胞肝細胞生長因子受體(HGFR)及 其調節之下游訊號分子的影響。 ................................................................. 37 圖九、構築pET-30a/GALNT1 質體。 ................................................................... 38 圖十、誘導表現GALNT1-His Tag 重組蛋白和純化。 ....................................... 39 圖十一、GALNT1 血清對於GALNT1-His Tag 重組蛋白具有特異性。 ........... 40 表一、siRNA 序列 ................................................................................................... 41 表二、即時定量聚合酶連鎖反應(Real-time Polymerase Chain Reaction, Realtime- PCR)引子(Primer)序列 ......................................................................... 42 表三、十二烷基硫酸鈉聚丙烯醯胺凝膠(Sodium dodecyl sulfate polyacrylamide gel)配法 ........................................................................................................... 43 表四、西方墨點法(Western blot analysis)所使用之抗體 ..................................... 44 表五、純化重組蛋白所使用溶液 ........................................................................... 45 | |
| dc.language.iso | zh-TW | |
| dc.subject | GALNT1 | zh_TW |
| dc.subject | 胰臟癌 | zh_TW |
| dc.subject | 醣化作用 | zh_TW |
| dc.subject | 肝細胞生長因子受體 | zh_TW |
| dc.subject | 多株抗體 | zh_TW |
| dc.subject | 胰臟癌 | zh_TW |
| dc.subject | 醣化作用 | zh_TW |
| dc.subject | GALNT1 | zh_TW |
| dc.subject | 肝細胞生長因子受體 | zh_TW |
| dc.subject | 多株抗體 | zh_TW |
| dc.subject | hepatocyte growth factor receptor | en |
| dc.subject | pancreatic cancer | en |
| dc.subject | polyclonal antibody | en |
| dc.subject | hepatocyte growth factor receptor | en |
| dc.subject | glycosylation | en |
| dc.subject | GALNT1 | en |
| dc.subject | GALNT1 | en |
| dc.subject | glycosylation | en |
| dc.subject | polyclonal antibody | en |
| dc.subject | pancreatic cancer | en |
| dc.title | 在 HPAC 胰臟癌細胞抑制 N-acetylgalactosaminyltransferase 1
的表現會抑制其惡性特性 | zh_TW |
| dc.title | Knockdown of N-acetylgalactosaminyltransferase 1 inhibits
malignant phenotypes of HPAC pancreatic cancer cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李建智,陳啟豪 | |
| dc.subject.keyword | 胰臟癌,醣化作用,GALNT1,肝細胞生長因子受體,多株抗體, | zh_TW |
| dc.subject.keyword | pancreatic cancer,glycosylation,GALNT1,hepatocyte growth factor receptor,polyclonal antibody, | en |
| dc.relation.page | 45 | |
| dc.identifier.doi | 10.6342/NTU201700487 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-02-10 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 解剖學暨細胞生物學科所 | |
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