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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周綠蘋(Lu-Ping Chow) | |
| dc.contributor.author | Fan-Ying Chiang | en |
| dc.contributor.author | 江芳瑩 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:56:26Z | - |
| dc.date.available | 2017-09-19 | |
| dc.date.copyright | 2012-09-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-18 | |
| dc.identifier.citation | 1. Kaku, T., et al., Histological classification of ovarian cancer. Med Electron Microsc, 2003. 36(1): p. 9-17.
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Oh, Unraveling insulin-like growth factor binding protein-3 actions in human disease. Endocr Rev, 2009. 30(5): p. 417-37. 50. Hayashida, Y., et al., Calreticulin represses E-cadherin gene expression in Madin-Darby canine kidney cells via Slug. J Biol Chem, 2006. 281(43): p. 32469-84. 51. Wu, C.Y., et al., IL-1beta induces MMP-9 expression via a Ca2+-dependent CaMKII/JNK/c-JUN cascade in rat brain astrocytes. Glia, 2009. 57(16): p. 1775-89. 52. Wang, H.H., H.L. Hsieh, and C.M. Yang, Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid. J Neuroinflammation, 2010. 7: p. 84. 53. Fong, Y., et al., Acacetin inhibits TPA-induced MMP-2 and u-PA expressions of human lung cancer cells through inactivating JNK signaling pathway and reducing binding activities of NF-kappaB and AP-1. J Food Sci, 2010. 75(1): p. H30-8. 54. Gerke, V. and S.E. Moss, Annexins: from structure to function. Physiol Rev, 2002. 82(2): p. 331-71. 55. Yi, M. and J.E. Schnitzer, Impaired tumor growth, metastasis, angiogenesis and wound healing in annexin A1-null mice. Proc Natl Acad Sci U S A, 2009. 106(42): p. 17886-91. 56. Kang, H., J. Ko, and S.W. Jang, The role of annexin A1 in expression of matrix metalloproteinase-9 and invasion of breast cancer cells. Biochem Biophys Res Commun, 2012. 57. Liu, Y.F., et al., Identification of annexin A1 as a proinvasive and prognostic factor for lung adenocarcinoma. Clin Exp Metastasis, 2011. 28(5): p. 413-25. 58. Healy, S., D.H. Khan, and J.R. Davie, Gene expression regulation through 14-3-3 interactions with histones and HDACs. Discov Med, 2011. 11(59): p. 349-58. 59. van Hemert, M.J., H.Y. Steensma, and G.P. van Heusden, 14-3-3 proteins: key regulators of cell division, signalling and apoptosis. Bioessays, 2001. 23(10): p. 936-46. 60. Ravi, D., et al., 14-3-3 sigma expression effects G2/M response to oxygen and correlates with ovarian cancer metastasis. PLoS One, 2011. 6(1): p. e15864. 61. Ghahary, A., et al., Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts. J Invest Dermatol, 2005. 124(1): p. 170-7. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65653 | - |
| dc.description.abstract | 卵巢癌是全球女性致死率最高之婦科癌症之一。大多數的惡性卵巢腫瘤為上皮細胞型態，並且大部分病患在被診斷出時都已經發生癌症轉移的現象。然而病人在接受手術切除及化療後，仍然具有很高的復發率，晚期病人平均存活期更是只有四年。卵巢上皮細胞癌 (epithelial ovarian cancers)，根據組織型態可以分為四大類：漿液型 (serous)、黏液型 (mucinous)、子宮內膜樣 (endometrioid) 和透明細胞型(clear cell)，其中子宮內膜樣約占其中10 ~ 20 %。實驗室先前從病患腫瘤組織中分離出一細胞株─OVTW59，並且運用Transwell assay將細胞株根據侵襲能力細分，原始分離出可少量穿過Transwell的細胞株命名為P0，能通過四次Transwell篩選的細胞株名命為P4。並且從先前微陣列 (cDNA microarray) 分析，發現抑癌基因insulin-like growth factor binding protein-3 (IGFBP-3) 於P0及P4細胞當中有差異表現，並且IGFBP-3的表現量與病人的預後、存活率呈現負相關。
為了釐清與癌症轉移相關之蛋白參與卵巢癌轉移的機制，於是我們以二維電泳將P0及P4細胞的蛋白體展開，並尋找其中差異表現的蛋白質，並以液相層析─質譜儀鑑定身分。再運用生物資訊軟體─Ingenuity Pathway Analysis (IPA) 建構P0及P4細胞差異表現之蛋白質的網絡，及分析其可能扮演的角色。從IPA的分析結果，我們選定與腫瘤細胞株侵襲性有關的Calreticulin做後續的研究。從西方墨點法(Western blotting) 的實驗結果發現Calreticulin的表現確實與卵巢癌細胞株的侵襲性呈現正相關。此外，我們藉由大量表現及抑制生成Calreticulin，證實Calreticulin的確會影響卵巢癌細胞株細胞遷移 (migration)、侵襲 (invasion) 及分泌基質金屬蛋白酶 (matrix metalloproteinase-2/9, MMP-2/9) 的能力。然而IGFBP-3、Calreticulin和MMP-2/9及與細胞遷移相關蛋白之間的調控機制，仍有待後續研究釐清。 | zh_TW |
| dc.description.abstract | Ovarian cancer is the most lethal gynecological cancer among women worldwide. The majority of malignant ovarian tumors are epithelial type and metastasis has occurred in most patients at the time of diagnosis. Subsequent recurrence rate is high after complete surgery and chemotherapy. Survival is at about 4 years for patients with advanced ovarian cancer.
Based on the morphological criteria, epithelial ovarian cancers (EOC) are classified into four histological subtypes: serous, mucinous, endometrioid and clear cell. The endometrioid carcinoma (EC) subtype represents about 10 to 20 % of EOC. In our previous study, we have established an ovarian endometrioid carcinoma cell line OVTW59. The original lines were designated as P0, and the highly invasive subline P4 was isolated from 4 rounds of invasion selection. By cDNA microarray analysis, we have demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) is an invasion suppressor gene. Patients with low serum IGFBP-3 level are correlated with poor survival. In this study, we want to identify metastasis-associated proteins from ovarian endometrioid carcinoma cell line OVTW59. We used 2D-electrophoresis to compare the protein profiles between the original cell line P0 and the highly invasive subline P4, and potential candidates were subsequently identified by LC-MS/MS. Then we built up a potential network involving those differentially expressed proteins by Ingenuity Pathway Analysis. We found a novel metastasis-associated protein, calreticulin. Western blotting demonstrated that calreticulin expression level is highly correlated with cancer invasion. By overexpressing and knocking down Calreticulin, we found that Calreticulin regulates cell migration, invasion, and MMP-2/9 secretion. However, the regulatory mechanism among IGFBP-3, Calreticulin, MMP-2/9, and cell migration-associated proteins remains to be further elucidated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:56:26Z (GMT). No. of bitstreams: 1 ntu-101-R99442030-1.pdf: 7912456 bytes, checksum: 7980ea60b8952d26c7591c246fcd0733 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 謝辭 i
摘要 ii Abstract iii 縮寫 v 第一章 導論 1 第一節 卵巢癌 (Ovarian cancer) 1 1.1卵巢癌之流行病學及分類 1 1.2卵巢癌的診斷、治療及預後 1 1.3人類卵巢癌細胞株 3 第二節 癌症侵襲及轉移 (Invasion and metastasis) 3 2.1癌症侵襲及轉移之機制 3 2.2與癌症侵襲及轉移相關之蛋白 4 2.3 Matrix metalloproteinases與癌症侵襲及轉移之關係 6 第三節 研究動機 8 第二章 實驗材料 9 第一節 卵巢癌細胞株 9 第二節 儀器及裝置 9 第三節 大腸桿菌及質體 10 第四節 酵素 11 第五節 抗體 11 第六節 試劑組與藥品 11 第七節 軟體與資料庫 13 第三章 實驗方法 14 第一節 卵巢癌細胞株的培養 14 1.1培養基 (medium) 的配置 14 1.2細胞的培養 14 1.3細胞的計數 14 第二節 條件培養液 (Conditioned medium) 15 2.1細胞的準備 (plating) 15 2.2條件培養液的收取及濃縮 15 第三節 蛋白質分析法 15 3.1蛋白質濃度測定 (660 nm Protein Assay) 15 3.2十二烷基磺酸鈉-聚丙烯醯胺膠體電泳分析 (SDS-PAGE) 16 3.3西方墨點法 (Western blotting) 17 第四節 二維電泳分析 18 4.1樣本處理 18 4.2第一維膠片 (IPG strip) 重新水合化 19 4.3第一維等電點展開 (isoelectric focusing, IEF) 19 4.4第二維電泳分析 (SDS-PAGE) 20 4.5染色與退染 20 第五節 膠體原位酵素切割及蛋白質鑑定 20 第六節 重組蛋白之建立 21 第七節 小髮夾RNA（shRNA）之建立 21 第八節 質體轉染 (Transfection) 21 第九節 細胞生存能力試驗 (MTT Assay) 22 9.1 MTT reagent 配置 22 9.2細胞準備 22 9.3 MTT活性偵測 22 第十節 細胞傷口癒合分析 (Wound healing assay) 23 第十一節 細胞侵襲能力試驗 (Transwell assay) 23 11.1 Transwell配置 23 11.2細胞準備 23 11.3細胞侵襲能力偵測 23 第十二節 細胞Matrix metalloproteinase分析 24 12.1細胞的準備 (plating) 24 12.2明膠脢普法 (Gelatin zymography) 24 12.3酵素免疫分析法 (ELISA) 25 第四章 結果 27 第一節 P0及P4細胞之特性 27 第二節 P0及P4細胞差異表現之蛋白 27 2.1二維電泳分析鑑定P0及P4細胞差異表現之蛋白 27 2.2 軟體分析差異表現蛋白之分子功能 28 2.3軟體建構差異表現蛋白之網絡及分析其與癌細胞轉移之關係 28 第三節 利用西方墨點法確認Calreticulin為癌細胞轉移相關之蛋白 30 第四節 P0及P4細胞於Matrix metalloproteinase具有差異表現 30 第五節 Calreticulin重組蛋白之建立及shRNA之篩選 30 第六節 Calreticulin之功能性分析 32 6.1 Calreticulin並未影響細胞生存能力 32 6.2 Calreticulin促進癌細胞移動 32 6.3 Calreticulin促進癌細胞侵襲 32 6.4 Calreticulin促進癌細胞分泌Matrix metalloproteinase 33 第七節 結論 34 第五章 討論 35 第一節 實驗方法學討論 35 1.1 二維電泳分析法的優點與限制 35 1.2 軟體預測之優點與限制 35 第二節 Calrecticulin於癌症進展 (cancer progression) 中扮演的角色 36 第三節 Matrix metalloproteinase於卵巢癌表現之情形 36 第四節 IGFBP-3、Calreticulin和MMP-2/9之間可能的調控機制 37 第五節 其他鑑定到之蛋白與癌症轉移的關係 38 第六節 結語與未來展望 39 6.1 Calreticulin促進卵巢癌細胞株的移動及侵襲能力 39 6.2 未來研究方向與展望 40 第六章 參考文獻 41 圖表 46 附錄 74 | |
| dc.language.iso | zh-TW | |
| dc.title | 運用蛋白質體學方法鑑定卵巢癌細胞株中與癌症轉移相關的蛋白 | zh_TW |
| dc.title | Identification of metastasis-associated proteins in ovarian cancer cell line by proteomics approach | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 童寶玲(Pao-Ling Torng),林欽塘(Chin-Tarng Lin),許麗卿(Lih-Ching Hsu) | |
| dc.subject.keyword | 卵巢癌,癌症轉移,Calreticulin,基質金屬蛋白酶, | zh_TW |
| dc.subject.keyword | Ovarian cancer,metastasis,Calreticulin,Matrix metalloproteinases, | en |
| dc.relation.page | 78 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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