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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳林祈 | |
| dc.contributor.author | Keng-Jung LEE | en |
| dc.contributor.author | 李庚融 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:24:08Z | - |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-06-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51033 | - |
| dc.description.abstract | 上皮黏蛋白1-羧基端（MUC1-COOH; MUC1-C）與Kruppel-like factor 4 (KLF4) 轉錄因子皆與癌細胞凋亡具有高度的相關性。PE21區域位於腫瘤抑制基因p53之啟動子（promoter）中；而MUC1-C 會與KLF4形成二聚體（MUC1-C/KLF4）鍵結至PE21區域進而抑制p53表現。本研究設計之誘導寡核苷酸（decoy oligonucleotide; dODNs）為雙股具有21個核苷酸，其序列由PE21區域演繹而得。本研究探討dODNs-5’與dODNs-3’兩種序列，鍵結在MUC1-C/KLF4複合體，去阻斷MUC1-C/KLF4複合體與PE21之間鍵結作用，進而活化p53基因，導致人類乳癌細胞（MCF-7）凋亡。在細胞凋亡實驗中，將兩組dODNs轉染至MCF-7 中分別使29.4%與24.5%的細胞凋亡。同時研究將 dODNs的序列隨機組合之新序列為負控制組（scr-1與scr-2），對照實驗顯示生存率依序為83.3%、81.5%。其中空白實驗組（blank）的生存率為81.1%。此外，在基因表現方面，可分別促進p53基因表現達2.99與2.72倍。進一步針對dODNs進行毒性測試，將dODNs轉染至人類纖維母細胞（fibroblast）生存率皆在90%以上。最後，我們也利用免疫染色法，顯示出兩組dODNs位於細胞核內，說明了本研究在驗證機制上，dODNs可進入細胞核抑制MUC1-C/KLF4複合體與PE21作用。由以上結果顯示，本研究提出一個策略以MUC1-C/KLF4複合體為標靶的dODNs，具有選擇性地使MCF-7凋亡。 | zh_TW |
| dc.description.abstract | The MUC1 COOH-terminal subunit (MUC1-C) and the Kruppel-like factor 4 (KLF4) transcription factor are both highly associated with cell apoptosis. The MUC1-C binds to KLF4 to form the MUC1-C/KLF4 complex, which represses p53 transcription by binding to the PE21 element within the tumor suppressor p53 promoter. In this work, we designed two 21-mer double-stranded decoy oligonucleotides (dODNs-5’ and dODNs-3’) based on the MUC1-C/KLF4 PE21 element. Blocking the interaction between MUC1-C/KLF4 and PE21 by the dODNss resulted in up-regulating p53 gene expression and inducing human breast cancer cell apoptosis. In apoptosis assay, transfection of the dODNs-5’ and the dODNs-3’ respectively resulted in 29.4% and 24.5% apoptosis. The viability of transfection of the negative control scrambled-sequence oligonucleotides (1-scr and 2-scr) and the blank control are 83.3%、81.5% and 81.1% sequentially. Both of the dODNs-5’ and dODNs-3’ up-regulated p53 gene expression 2.99 and 2.72 times, respectively. Besides, we chose the fibroblast cells served as a normal cell control. In the fibroblast cells, transfection of the dODNss and the SCRs showed that the viability were all at least 90% in the apoptosis assay. We also showed that the dODNss interacted with MUC1-C/KLF4 in nucleus by immunofluorescence assay. In comparison, the SCRs localize at the outside of the nucleus. To conclude, inhibition of the MUC1-C / KLF4 complex by using a transcription factor decoy can induce human breast cancer cell apoptosis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:24:08Z (GMT). No. of bitstreams: 1 ntu-105-R02631043-1.pdf: 29400541 bytes, checksum: 1fe4b51dc1c5943803acf81f0d8c376e (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 i
目錄 v 圖目錄 vii 表目錄 ix 第一章 緒論 1 1.1前言 1 1.2研究動機 3 1.3研究目的 5 1.4研究架構 7 第二章 文獻探討 10 2.1啟動子技術（Promoter Technique）應用於癌症療法 10 2.2 MUC1-C致癌蛋白介紹與標靶 MUC1-C的策略 12 2.2.1 MUC1-C與乳癌的關係 12 2.1.2標靶MUC1的策略 17 2.3 KLF4介紹與癌症的關係 20 2.4 p53與癌症的關係 23 2.4.1 p53的功能 24 2.5生物分子嵌合 25 第三章 材料與實驗方法 26 3.1本研究使用之材料與儀器 26 3.1.1實驗使用的藥品 26 3.1.2本研究所使用到的儀器 28 3.1.3 dODNs與核酸引子 30 3.2實驗步驟 32 3.2.1細胞培養 32 3.2.2細胞繼代培養 32 3.2.3細胞計數 32 3.2.4轉染誘導寡核苷酸 33 3.2.5細胞存活率分析 33 3.2.6 ANNEXIN-V/PI雙染法 34 3.2.7西方點墨法檢測基因水準 34 3.2.8 PI染色檢定細胞週期 37 3.2.9免疫染色法 37 第四章 結果與討論 39 4.1誘導寡核苷酸之轉染效率 39 4.1.1 dODNss與SCRs對人類乳癌細胞轉染之轉染效率比較 40 4.1.2誘導寡核苷酸轉染至MCF-7與fibroblast之轉染效率比較 43 4.2誘導寡核苷酸對於人類乳癌細胞之影響 46 4.2.1誘導寡核苷酸對於人類乳癌細胞細胞凋亡之型態 46 4.2.2誘導寡核苷酸對於人類乳癌細胞活性之影響...................................................50 4.2.3誘導寡核苷酸誘導人類乳癌細胞程序性凋亡 52 4.2.4誘導寡核苷酸對於人類纖維母細胞細胞週期之影響..................................... 56 4.3誘導寡核苷酸對於人類纖維母細胞之影響 58 4.3.1誘導寡核苷酸對於人類纖維母細胞生存率之影響 59 4.3.2誘導寡核苷酸對於人類纖維母細胞程序性凋亡之分析 61 4.3.3誘導寡核苷酸對於人類纖維母細胞細胞週期之影響 64 4.4誘導寡核苷酸對於人類乳癌細胞促進腫瘤抑制基因p53 66 4.5利用電腦輔助嵌合軟體檢視 68 4.5.1以3D-DART預測誘導寡核苷酸之雙股結構 68 4.5.2利用I-TASSER 預測MUC1-C的三維結構 69 4.5.3誘導寡核苷酸與MUC1-C嵌合的結果 70 4.6免疫染色檢視誘導寡核苷酸與MUC1-C交互作用 71 第五章 結論與未來展望 74 5.1結果摘要 74 5.2結論 77 5.3未來展望 78 參考文獻 79 附錄 88 附錄一 人類乳癌細胞與人類纖維母細胞之細胞型態 88 附錄二 以免疫染色檢視KLF4 在細胞中的位置 90 | |
| dc.language.iso | zh-TW | |
| dc.subject | breast cancer | en |
| dc.subject | Kruppel-like factor 4（KLF4) | en |
| dc.subject | Kruppel-like factor 4（KLF4) | en |
| dc.subject | p53 tumor suppressor gene | en |
| dc.subject | MUC1 COOH-terminal subunit (MUC1-C) | en |
| dc.subject | MUC1 COOH-terminal subunit (MUC1-C) | en |
| dc.subject | p53 tumor suppressor gene | en |
| dc.subject | decoy oligonucleotide | en |
| dc.subject | breast cancer | en |
| dc.subject | decoy oligonucleotide | en |
| dc.title | 以誘餌寡核苷酸抑制MUC1-C / KLF4複合體
與誘導人類乳癌細胞凋亡之研究 | zh_TW |
| dc.title | On the Inhibition of MUC1-C and KLF4 Complex for Inducing Human Breast Cancer Cell Apoptosis Using a Decoy Oligonucleotide | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳倩瑜,廖泰慶,侯詠德 | |
| dc.subject.keyword | 上皮黏蛋白1-羧基端(MUC1-COOH,MUC1-C),Kruppel-like factor 4(KLF4),腫瘤抑制基因p53,誘導寡核?酸,乳癌, | zh_TW |
| dc.subject.keyword | MUC1 COOH-terminal subunit (MUC1-C),Kruppel-like factor 4（KLF4),p53 tumor suppressor gene,decoy oligonucleotide,breast cancer, | en |
| dc.relation.page | 93 | |
| dc.identifier.doi | 10.6342/NTU201600411 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-06-21 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物產業機電工程學研究所 | zh_TW |
| Appears in Collections: | 生物機電工程學系 | |
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| ntu-105-1.pdf Restricted Access | 28.71 MB | Adobe PDF |
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