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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林欽塘(Chin-Tarng Lin) | |
dc.contributor.author | Yu-Chyi Hwang | en |
dc.contributor.author | 黃鈺琪 | zh_TW |
dc.date.accessioned | 2021-06-15T00:17:57Z | - |
dc.date.available | 2014-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-04-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41391 | - |
dc.description.abstract | 鼻咽癌是中國南方、新加坡、香港、台灣等地的中國人好發的癌症。然而,它的病源因子尚未完全被研究清楚。目前被認為EB病毒 (Epstein-Barr virus)很可能和鼻咽癌有密切關係。然而,EB病毒和它的宿主鼻咽癌的基因之間所牽涉到的分子機轉仍然不很明確。為了釐清引起鼻咽癌而改變的基因表現,我們設計了兩組全面性的基因篩選的實驗來鑑定可能參與鼻咽癌細胞變化的基因。
第一個是兩組互相比較的基因晶片實驗:一組是比較鼻咽癌(無EB病毒)和正常上皮細胞的基因晶片,另一組是比較有EB病毒感染和沒有EB病毒感染的鼻咽癌基因晶片。我們利用交叉比較這兩組基因晶片所得到的資料而鑑定它們彼此間的相關性。結果顯示EB病毒似乎比較傾向於調節在鼻咽癌細胞中已經有差異表現的基因,對於無異於正常細胞表現的基因則沒有什麼調控現象。綜合這兩組基因的資料可以顯示利用全面性的基因體掃瞄方式研究鼻咽癌以及EB病毒感染的鼻咽癌細胞的轉錄基因量,可以成功的呈現EB病毒感染具有調控有差異表現的鼻咽癌基因的能力。 第二個實驗是結合PCR-Select™ cDNA 相減雜交(subtractive hybridization) 技術和基因晶片分析來鑑定鼻咽癌細胞株和正常鼻咽細胞之間的差異基因表現。我們發現在大部份的鼻咽癌細胞株和病人檢體中發現NOLC1基因的表現比正常細胞高出許多。利用NOLC1基因的干擾RNA質體(shRNA)轉染鼻咽癌細胞的實驗顯示減少NOLC1基因表現會降低鼻咽癌細胞的生長速率。在帶有異種移植(xenografts)鼻咽癌細胞的嚴重混合性免疫缺乏症(severe combined immune deficiency,簡稱SCID)老鼠的實驗中顯示:減少NOLC1基因表現的shRNA轉染鼻咽癌細胞在SCID老鼠體內第十一週時,腫瘤大小比控制組小了百分之八十二,而且腫瘤內呈現出嚴重的細胞凋亡(apoptosis)。在檢測與細胞生長、細胞凋亡、和血管新生相關的基因表現後,發現轉染NOLC1基因shRNA的鼻咽癌細胞內,MDM2、MMP9、VEGF等基因表現降低。相反的,TNF、BAX、CASP1等基因的表現被調節後上升。在染色體免疫沉澱(Chromatin Immunoprecipitation) 和共同轉染的實驗中顯示TP53蛋白質調控MDM2基因的表現需要NOLC1蛋白質的共同活化。以上這些發現顯示NOLC1在鼻咽癌細胞中扮演一個促進轉錄調控的角色,進而調控與細胞凋亡和細胞生長相關的基因,並且證明了NOLC1蛋白質與TP53蛋白質在鼻咽癌細胞中合力活化MDM2基因的啟動子(promoter)。 | zh_TW |
dc.description.abstract | Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. Its etiological factors are not yet well defined. It was proposed that Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms involved in the effect of EBV on NPC host genes remained vague to date. To clarify the possible genetic alteration, we used two approaches to globally screen for genes involved in NPC.
The first one was designed by two sets of microarray experiments; NPC (EBV-free) compared with normal epithelial cells and EBV+ compared with EBV-free NPC arrays. We analyzed the datasets by cross-comparison gene clusters involved in EBV targeting and the NPC host gene expression profiles to identify the correlation between them. Result from the statistical analysis showed that EBV regulates genes from the differentially expressed group in NPC cells than those from the unchanged expression group. Taken together, the genome-wide comparative scanning of transcription levels of genes in EBV+ and EBV- of NPC has successfully demonstrated that EBV infection transduces signals involved in NPC gene expression. The second approach was combined the PCR-Select™ cDNA subtractive hybridization with microarray analysis to identify differential gene expression between NPC cell lines and normal nasomucosal cells. We identified that human NOLC1 (nucleolar and coiled-body phosphoprotein 1) gene expression was upregulated in most NPC cell lines and biopsy specimens. NOLC1 knockdown NPC cells showed reduced cell proliferation rates. In SCID mice bearing NPC xenografts derived from these transfected cell lines, the NPC xenograft tumors had diminished about 82% in size with marked tumor cell apoptosis compared with the control at week 11. To understand the contribution of NOLC1 in controlling tumor cell proliferation, a screening was performed to measure the expression of 26 genes related to apoptosis and angiogenesis in the transfectants showed that MDM2, MMP9, and VEGF expression were downregulated, whereas the expression of TNF, BAX, and CASP1 was upregulated connection. Chromatin immunoprecipitation and cotransfection experiments showed that TP53-regulated MDM2 expression requires NOLC1 coactivation. These findings suggest that NOLC1 plays a role as a transcriptional regulator to regulate apoptosis-related and proliferation-related genes, and demonstrate a relationship between NOLC1 and TP53 in the synergistic activation of the MDM2 promoter in NPC cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:17:57Z (GMT). No. of bitstreams: 1 ntu-98-D89444002-1.pdf: 6878473 bytes, checksum: d4a19b0f9309321aa69e8edbb8b3d084 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 口試委員審訂書 i
誌謝 Acknowledgement ii 中文摘要 iii Abstract v 目錄 Table of Contents vii List of Figures ix List of Tables xi 1. Introduction 1 1.1 Nasopharyngeal carcinoma (NPC) 1 1.2 EBV in NPC 3 1.3 NOLC1 gene 9 1.4 MDM2 gene 13 2. Materials and Methods 17 2.1 Biological samples 17 2.2 EBV Infection 19 2.3 Immunocytochemistry 21 2.4 In vitro scratch wound healing assay 23 2.5 MTT Assays 23 2.6 Preparation of RNA 24 2.7 Global genes screening 26 2.8 Quantitative Real-time RT-PCR (qRT-PCR) 35 2.9 Suppression subtractive hybridization (SSH) 40 2.10 Nylon cDNA microarray analysis and screening of the cDNA library with subtracted probes 41 2.11 Western blotting 42 2.12 Immunohistochemical staining 43 2.13 Establishment of stable shRNA transfectants 44 2.14 Tumor growth in SCID mice 45 2.15 Touchdown RT-PCR 45 2.16 Gene regulation assay 46 2.17 Statistical analysis 49 3. Results 50 3.1 EBV infection to NPC cells can enhance the host cell proliferation and migration rate 50 3.2 Microarray analysis of differential gene expressions in NPC 51 3.3 Verification of Selected Genes by qRT-PCR 54 3.4 Genes selected by suppression subtractive hybridization and microarray 56 3.5 NOLC1 gene expression is upregulated in most NPC tumor cells 57 3.6 NOLC1 is highly expressed in the NPC biopsy specimens 58 3.7 Inhibition of NOLC1 expression decreases proliferation and increases apoptosis in NPC cells 58 3.8 NOLC1 regulates MDM2 gene expression. 60 3.9 NOLC1 acts synergistically with TP53 to upregulate MDM2 expression 61 3.10 NOLC1 interacts with the MDM2 promoter at the TP53 binding site 64 4. Discussion 66 5. Figures 75 6. Tables 101 7. References 114 8. Appendex 131 | |
dc.language.iso | en | |
dc.title | 鼻咽癌基因NOLC1 調控腫瘤生長之機轉 | zh_TW |
dc.title | Mechanism of NOLC1 gene regulating nasopharyngeal carcinoma progression | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 吳漢忠(Han-Chung Wu) | |
dc.contributor.oralexamcommittee | 黃奇英(Chi-Ying Huang),葉寧馨(Ning-Hsing Yeh),林淑華(Shu-Wha Lin),王萬波(Won-Bo Wang),陳美如(Mei-Ru Chen),林中梧(Chung-Wu Lin),高承福(Cheng-Fu Kao) | |
dc.subject.keyword | EB病毒,鼻咽癌,基因晶片,相減雜交,異種移植,染色體免疫沉澱,細胞凋亡,NOLC1,TP53,MDM2, | zh_TW |
dc.subject.keyword | Epstein–Barr virus (EBV),Nasopharyngeal carcinoma (NPC),microarray,xenografts,Chromatin immunoprecipitation,apoptosis,NOLC1,TP53,MDM2, | en |
dc.relation.page | 132 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-04-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 病理學研究所 | zh_TW |
顯示於系所單位: | 病理學科所 |
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