DC 欄位 | 值 | 語言 |
dc.contributor | 楊雅倩 | zh-TW |
dc.contributor | Yang, Ya-Chien | en |
dc.contributor | 臺灣大學:醫學檢驗暨生物技術學研究所 | zh-TW |
dc.contributor.author | 張嘉芸 | zh-TW |
dc.contributor.author | Chang, Chia-Yun | en |
dc.creator | 張嘉芸 | zh-TW |
dc.creator | Chang, Chia-Yun | en |
dc.date | 2008 | en |
dc.date.accessioned | 2010-05-11T07:50:56Z | - |
dc.date.accessioned | 2018-07-06T08:01:44Z | - |
dc.date.available | 2010-05-11T07:50:56Z | - |
dc.date.available | 2018-07-06T08:01:44Z | - |
dc.date.issued | 2008 | - |
dc.identifier.other | U0001-2407200820171800 | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/182951 | - |
dc.description.abstract | 大腸直腸癌 (colorectal cancer, CRC)是常見的癌症,為國人癌症十大死因的第三名,偶發性CRC發生過程大多會從腺瘤變成腺癌,且又依照不同的基因轉變分為LOH (loss of heterozygosity)及MSI (microsatellite instability)兩種路徑。除了基因序列變異的累積會造成癌細胞的發展,基因後生遺傳修飾 (epigenetic modification)也扮演重要角色。常見的基因後生遺傳修飾調控即藉由DNA methyltransferases (DNMTs)將基因啟動子 (promoter) CpG位點之cytosine第5號碳進行甲基化 (mC)。CpG甲基化會抑制啟動子和轉錄因子 (transcription factors, TFs)的結合,使基因無法轉錄。目前CRC後生遺傳修飾的研究上,已發現許多抑癌基因受到DNA甲基化抑制的調控。本研究室於尋找CRC相關抑癌基因的研究中,分析腫瘤在第四號染色體微衛星標記發生LOH的情形,結果於4q25-4q28.3發現三個LOH發生率高的區域,而細胞吸附分子protocadherin 10 (PCDH10)基因即位於其中一個區域上。近期有研究指出PCDH10可能是抑癌基因,在鼻咽癌、血癌或其他腫瘤中,此基因的表現似乎受到後生遺傳修飾的抑制。為了探討大腸直腸癌中PCDH10基因是否受到啟動子過度甲基化 (promoter hypermethylation)的調控,本論文利用一系列DNA甲基化分析方法,檢測大腸直腸癌細胞株及CRC病人檢體PCDH10甲基化情形,並進一步統計PCDH10甲基化與臨床資料及其他癌症相關基因甲基化的相關性。結果顯示,CRC腫瘤之PCDH10甲基化程度明顯高於大腸正常黏膜組織,並具有統計上顯著差異,而腫瘤之PCDH10甲基化於病患年齡、性別、腫瘤位置、腫瘤分化程度及CRC分期則無顯著差異。此外,藉由DNA去甲基化藥物5-aza-2''-deoxycytidine (5-Aza-CdR)處理CRC細胞株SW620,可使PCDH10基因啟動子去甲基化並恢復其mRNA的表現,進一步證明在大腸直腸癌中,PCDH10基因的表現確實受到DNA甲基化的調控。 | zh-TW |
dc.description.abstract | Colorectal cancer (CRC) is one of the most common malignancies and is the third leading cause of cancer death in Taiwan. Most sporadic CRCs are thought to develop from benign adenomas to carcinoma. The two major pathways in colorectal carcinogenesis, LOH (loss of heterozygosity) and MSI (microsatellite instability), accumulate different genetic abnormalities. Tumorigenesis is not only linked to genetic alteration as a contribution but also to epigenetic regulation as a major player in cancer development. DNA methylation remains the best-studied epigenetic mechanism. Methylation at the C-5 position of cytosine residues present in CpG dinucleotides by DNA methyltransferases (DNMTs). This methylation inhibits gene expression by directly blocking the binding of transcription factors (TFs) to the target gene promoter sequence. Tumor suppressor genes (TSGs) silencing by DNA methylation has been found in many cases of cancer. In our preliminary study of LOH frequency on chromosome 4 in cases of CRC, three LOH loci were frequently defined around 4q25-4q28.3. A cell adhesion molecule, protocadherin 10 (PCDH10), is located in one of these regions. In a recent study, PCDH10 was identified as a candidate TSG due to frequent silencing in nasopharyngeal, esophageal, lymphoma and multiple other carcinomas by DNA methylation. However, there is little known about PCDH10 methylation in CRC. Therefore, the purpose of this study was to deremine whether PCDH10 expression is regulated by DNA methylation. We analyzed the frequency of PCDH10 methylation in CRC cell lines and patient tissue samples. Our studies revealed that PCDH10 was highly methylated in both CRC cell lines and CRC tissues. Methylation of PCDH10 between tumor tissues of CRC patients and adjacent normal mucosa was significant, but was not necessarily correlated to age, sex, tumor location, tumor differentiation, or Dukes'' stage. On the other hand, the colon cancer cell, SW620, once treated with an epigenetic demethylation drug, 5-aza-2''-deoxycytidine (5-Aza-CdR), can restore PCDH10 RNA expression. Thus, PCDH10 expression is regulated by epigenetic modification and is frequently methylated in CRC. | en |
dc.description.tableofcontents | 致謝 i文摘要 ii 文摘要 iii一章 緒論 1一節 大腸直腸癌簡介 1. 大腸直腸癌 1. 大腸直腸癌的分期 3二節 基因後生遺傳修飾作用 5. 基因後生遺傳調控 5. 基因後生遺傳修飾藥物 7. 研究基因後生遺傳修飾的方法 8三節 Protocadherin 10 10. Cadherin superfamily 10. Protocadherin 10. PCDH10 11四節 研究目的 12二章 材料與方法 13一節 實驗材料 13. 細胞株 13. 細胞株培養 13. 大腸直腸組織 13. 健康者周邊血液 14二節 實驗方法 14. 抽取細胞株DNA 14. 抽取組織DNA 14. 抽取周邊血液DNA 15. 抽取細胞株RNA 15. 反轉錄-聚合酶連鎖反應 16. DNA及RNA濃度測量 16. DNA亞硫酸鈉處理 16. 甲基化特異性聚合酶連鎖反應 17. 亞硫酸鹽定序聚合酶連鎖反應 170. 結合亞硫酸鹽處理及限制酶分析法 181. DAPK1、hMLH1 及 PCDH10 基因甲基化之定義 192. 細胞增生及活性測試 193. 測試5-Aza-CdR處理對SW620之PCDH10基因去甲基化及RNA表現的影響 21三節 統計分析 21三章 實驗結果 22一節 DAPK1基因甲基化分析之結果 22二節 hMLH1 基因甲基化分析之結果 22三節 PCDH10 基因甲基化分析之結果 23四節 大腸直腸癌檢體基因甲基化與臨床資料的統計分析 25五節 大腸直腸癌細胞PCDH10基因mRNA的表現 29六節 以DNA去甲基化藥物處理大腸直腸癌細胞後對PCDH10基因表現的影響 30四章 討論 32 36 61考文獻 73圖 80表 82目錄igure 1. Methylation status of Death-associated protein kinase 1 (DAPK1) CpG island in cell lines and colorectal cancer (CRC) tissues 36igure 2. Methylation status of human mutL homolog 1 (hMLH1) CpG island in cell lines and colorectal cancer (CRC) tissues 37igure 3. Methylation status of Protocadherin10 (PCDH10) CpG island in cell lines, peripheral blood mononuclear cells (PBMCs) and colorectal tissues 38igure 4. Combined bisulfite restriction analysis of Protocadherin10 (PCDH10) CpG island in cell lines, peripheral blood mononuclear cells (PBMC) and CRC tissues 40igure 5. Bisulfite sequence analysis of Protocadherin10 (PCDH10) promoter CpG island in cell lines, peripheral blood mononuclear cells (PBMC) and colorectal tissues. 41igure 6. Promoter methylation and RNA expression of Protocadherin10 (PCDH10) in cell lines, peripheral blood mononuclear cells (PBMCs) and colorectal tissues 44igure 7. Methylation frequency of Protocadherin10 (PCDH10) at each CpG site in 12 CRC cell lines, 2 peripheral blood mononuclear cells (PBMCs) and 7 pairs of CRC tissues 45igure 8. Clinicopathological correlation of DAPK1、hMLH1 and PCDH10 gene methylation with age in CRC by regression analysis 46igure 9. Clinicopathological correlation of DAPK1、hMLH1 and PCDH10 gene methylation with age in CRC by unpair t-test 47igure 10. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation between normal mucosa and tumors in CRC 48igure 11. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation with gender in CRC 49igure 12. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation with tumor location in CRC 50igure 13. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation with tumor differentiation in CRC 51igure 14. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation with Dukes'' stage in CRC 52igure 15. Clinicopathological correlation of hMLH1 and PCDH10 gene methylation with disease-free survival in CRC tumors 53igure 16. Clinicopathological correlation of hMLH1 and PCDH10 methylation with overall survival in CRC tumors 54igure 17. PCDH10 mRNA expression in cell lines and CRC normal mucosa 55igure 18. Cell proliferation assay of SW480 and SW620 56igure 19. Cytotoxicity assay of 5-aza-2’-deoxycytidine (5-Aza-CdR) on SW480 and SW620 57igure 20. DNA demethylation and restored mRNA expression of Protocadherin10 (PCDH10) by 5-aza-2’-deoxycytidine (5-Aza-CdR) treatment 58目錄able 1. Cell line culture condition 61able 2. PCR primers used 63able 3. PCDH10 RT-PCR condition 64able 4. GAPDH RT-PCR condition 65able 5. DAPK1 MSP condition 66able 6. hMLH1 MSP condition 67able 7. PCDH10 MSP condition 68able 8. PCDH10 BSP condition 69able 9. PCDH10 COBRA condition 70able 10. Clinicopathological charateristics of CRC patients 71able 11. Clinicopathological correlation of DAPK1、hMLH1 and PCDH10 methylation in CRC 72 | en |
dc.format | application/pdf | en |
dc.format.extent | 2940816 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | zh-TW | en |
dc.language.iso | en_US | - |
dc.subject | 大腸直腸癌 | zh-TW |
dc.subject | PCDH10 | zh-TW |
dc.subject | 後生遺傳修飾 | zh-TW |
dc.subject | DNA甲基化 | zh-TW |
dc.subject | Colorectal cancer | en |
dc.subject | Epigenetic modification | en |
dc.subject | DNA methylation | en |
dc.subject.classification | [SDGs]SDG3 | - |
dc.title | 大腸直腸癌之PCDH10基因後生遺傳的研究 | zh-TW |
dc.title | Epigenetic Study of PCDH10 Gene in Colorectal Cancer | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/182951/1/ntu-97-R95424003-1.pdf | - |
item.fulltext | with fulltext | - |
item.grantfulltext | open | - |
item.languageiso639-1 | en_US | - |
顯示於: | 醫學檢驗暨生物技術學系
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