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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Tzu-Ying Lee | en |
| dc.contributor.author | 李姿瑩 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:49:27Z | - |
| dc.date.available | 2018-06-26 | |
| dc.date.copyright | 2008-07-07 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2008-06-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37890 | - |
| dc.description.abstract | 微型核醣核酸microRNA (miRNA) 是一群非常短的RNA，大約只有二十幾個鹼基，但卻具有影響基因表現的能力或蛋白質的生合成。miRNA與siRNA在生化機制及功能是相當類似的。很多證據指出miRNA能調控大多能轉錄出蛋白質的基因，因此，miRNA對於許多的細胞生理功能都扮演重大的調控，包括發育、細胞的增生與分化、細胞凋亡或癌症的生成與發展。近年來，約有五百多個miRNA基因被預測或透過實驗方式所驗證出來。由於目前推論出約大於一千多個miRNA基因存在人類基因組當中，而如何去發現或證實新的miRNA基因更是研究上的一大課題。李文雄院士以及施純傑博士的研究團隊發展透過組織作篩選的電腦分析的方式來預測新的miRNA基因及其調控的下游基因，因此我們能藉由實驗的方法在三種細胞株中作細胞驗證，分別是MCF-7乳癌細胞株、MCF-10A正常乳腺細胞、IMR-90肺部正常纖維母細胞，我們確認其中六種新的miRNAs存在於此三種細胞中，並進一步挑選其中兩種miRNA來作功能性及演化保留性分析，並利用冷光酶測定分析以及西方墨點法來驗證此新 miRNA的確存在並可以調控其下游基因的表現。 | zh_TW |
| dc.description.abstract | MicroRNAs are endogenous RNAs of 20-23 nucleotides in length that negatively regulate the expression of target genes by binding to the 3’-untranslated regions (UTRs) of the target mRNAs. The importance of miRNAs in animals is highlighted by the facts that they regulate a large proportion of protein-coding genes, and are involved in the control of a variety of processes including development, cell proliferation, tissue differentiation, apoptosis and metabolism. In addition, microRNAs are shown to be directly involved in cancer initiation and progression. Recent studies suggest that up to one third of human genes are regulated by miRNAs. However, although many human miRNA genes have been identified or predicted, it is believed that many more remain to be discovered. In this work, we identified the existence and measured the endogenous expression levels of novel miRNAs predicted by Dr. Wen-Hsiung Li and Dr. Arthur Chun-Chieh Shih; furthermore, we identified their target gene in different kinds of cell lines such as human breast cancer cell line MCF-7, non-tumorigenic MCF-10A and human lung fibroblasts IMR-90. In addition, we analyzed and verified the functions of the novel miRNAs using their mimics and inhibitors by luciferase reporter assay system and western blotting. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:49:27Z (GMT). No. of bitstreams: 1 ntu-96-R95b43003-1.pdf: 7462212 bytes, checksum: c5f542d2d41551a88e2fa36f613bf2ec (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 學位論文口試委員會審定書 II
誌謝 III 中文摘要 V Abstract VI 縮寫表 VII 第一章 前言 1 1-1 miRNAs的介紹 1 1-2 miRNA 與siRNA 的差異 2 1-3 miRNAs對於細胞的影響 3 1-4 miRNAs及其標靶基因的預測 4 1-5研究目標 4 第二章 實驗材料與方法 6 2-1 細胞培養 6 2-2 細胞總核糖核酸(含微型核糖核酸)萃取 10 2-3反轉錄反應 (Reverse Transcription) 11 2-3-1 miRNAs表現使用 11 2-3-2基因表現使用 12 2-4、即時定量聚合酶鏈鎖反應 (Real-Time PCR) 13 2-5 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 15 2-6 膠體電泳(DNA Gel Electrophrosis) 16 2-6-1洋菜膠體製備 16 2-6-2聚丙烯醯胺膠體製備 17 2-6-3膠體電泳分析 18 2-6-4膠體回收 18 2-7 小量質體萃取 (Plasmid Extraction) 20 2-8 TA cloning 21 2-9 菌落PCR 篩選 (Colony PCR Screening) 22 2-10 pMIR-REPORT-LAMB3 3’-UTR expression vector質體構築 24 2-11 pMIR-REPORT-P-2 target sequence expression vector質體構築 26 2-12 冷光酶活性測定分析 (Luciferase Reporter Assay) 28 2-12-1 miRNAs轉殖與報告基因載體轉殖 28 2-12-2 Luciferase與β-galactosidase訊號偵測 29 2-13 西方墨點法 (Westen Blotting) 30 2-13-1 SDS聚丙烯醯胺膠體製備 30 2-13-2 SDS聚丙烯醯胺膠體電泳分析 31 2-13-3 膠體電泳轉漬 32 2-13-4 抗體作用呈色分析 33 第三章 結果 34 3.1 利用PCR、電泳及cloning方式確認細胞中的確具有小片段RNA序列 34 3-2 新miRNAs及其調控之標靶基因在物種演化上具有序列保留性 35 3-3 利用real-time PCR來驗證miRNA及其標靶基因在轉殖後的基因表現量 36 3-4 利用冷光酶活性測定分析驗證新 miRNA-P-2 和 P-47-5p 能分別調控至其標靶基因 CREB3L3 和 LAMB3 37 3-4-1 miRNA-P-2與其標靶基因 CREB3L3 37 3-4-2 miRNA-P-47-5p與其標靶基因 LAMB3 38 3-5 利用西方墨點法來分析miRNA及其標靶基因的蛋白質產物表現量 39 第四章 討論 40 第五章 未來展望 42 圖與表 43 參考文獻 71 附錄 78 | |
| dc.language.iso | zh-TW | |
| dc.subject | 冷光酶 | zh_TW |
| dc.subject | 微型核醣核酸 | zh_TW |
| dc.subject | 調控基因 | zh_TW |
| dc.subject | 組織篩選 | zh_TW |
| dc.subject | 活性分析 | zh_TW |
| dc.subject | target gene | en |
| dc.subject | luciferase assay | en |
| dc.subject | tissue-selective | en |
| dc.subject | microRNA | en |
| dc.title | 鑑定人類新微型核醣核酸及其調控基因 | zh_TW |
| dc.title | Identification of Human Novel microRNAs and Their Targets | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李文雄(Wen-Hsiung Li),施純傑(Arthur Chun-Chieh Shih),黃宣誠(Hsuan-Cheng Huang) | |
| dc.subject.keyword | 微型核醣核酸,調控基因,組織篩選,冷光酶,活性分析, | zh_TW |
| dc.subject.keyword | microRNA,target gene,tissue-selective,luciferase assay, | en |
| dc.relation.page | 97 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-06-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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