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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 顏伯勳(Bo-Shiun Yen) | |
| dc.contributor.author | Jia-En Liu | en |
| dc.contributor.author | 劉嘉恩 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:21:20Z | - |
| dc.date.available | 2022-02-15 | |
| dc.date.available | 2022-11-24T03:21:20Z | - |
| dc.date.copyright | 2022-02-15 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-02-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80905 | - |
| dc.description.abstract | 報導基因是一組具有易於觀察、測量和量化之表現型的基因。它們應該是高度靈敏、方便和可靠的，並能快速檢測到結果。報導基因通常會連接在調控基因的啟動子附近，替換外顯子，並測量未知 DNA 序列調控轉錄的能力。內含報導基因的質體一般以轉染的方式送入細胞內。然而，免疫細胞只能透過病毒轉導方式獲得質體。由於病毒感染免疫細胞的困難，沒有太多關於使用免疫細胞進行報導基因檢測的研究。在我們之前的研究中，我們建立了一個螢光素酶報導基因檢測系統，成功地測量了 HEK293T 細胞中 NF-kB 啟動子驅動的轉錄活性；然而，相同的系統在 CCL2 啟動子上無法作用。我們相信對趨化素啟動子的調節作用在免疫細胞中會更加明顯。因此，本研究在單核球細胞中建立了功能性報導基因檢測系統。使用慢病毒載體，將編碼紅色螢光蛋白和帶有NF-kB啟動子的螢光素酶之基因轉導到THP-1單核球細胞中，並進一步培養出表達紅色螢光蛋白的轉導細胞。用 TNFa 和 IFNr 處理成功地誘導了 NF-kB 啟動子驅動的螢光素酶報導基因的表現，並且在doxycycline誘導的 SP110b 表現後，該報導基因的表現增加了約 17 倍。該結果證明本研究創建的報導基因檢測系統可用於單核球細胞，也可用於其他細胞。此外，在載體中插入多個限制酶切位點的接頭序列有助於將各種啟動子替換到載體中，使其成為測試單核球細胞或其他細胞群中目標啟動子活性非常有用的平台。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:21:20Z (GMT). No. of bitstreams: 1 U0001-0502202223070400.pdf: 5594636 bytes, checksum: 1e372ab4cb256957e3045ad55cae62c1 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | Acknowledgments……………………………………………………………………...i 摘要……………………………………………………………………………………ii Abstract……………………………………………………………………………….iv Contents……………………………………………………………………………….v Figure contents…………………………………………………………………….…viii Table contents………………………………..……………………………………...ix Chapter 1 Introduction…………………………………………………………………1 1.1 Reporter gene system…………………………………….…………..………1 1.1.1 Fluorescent protein reporter gene system…………...………………...…2 1.1.2 Luc reporter gene system…………………………………………..……2 1.1.3 Ruc reporter gene system…………………………………………..……3 1.2 SP110b………………………………………………………………….……3 1.3 Nuclear Factor kappa B………………………………………………………5 1.4 Chemokine…………………………………………………………...………6 1.5 Specific aim…………………………………………………………..………8 Chapter 2 Materials and methods…………………………………………….………..9 2.1 Cell Culture………………………………………………………..…………9 2.2 Preparation of Competent Cell……………………………………….………9 2.3 DNA Transformation………………………………………………….……10 2.4 DNA Extraction…………………………………………………………..…10 2.5 Gel Electrophoresis…………………………………………………………11 2.6 Transfection…………………………………………………………………12 2.7 Generation of Lentivirus……………………………………………………12 2.8 Lentivirus Transduction……………………………………………….……13 2.9 Fluorescence Assay…………………………………………………………14 2.10 Luciferase Reporter Assay………………………………………...………14 2.11 Polymerase chain reaction………………………………………………...15 Chapter 3 Results 3.1 Constructing pHAGE-TRE-DsRed-NFkB-Luc plasmid……………………16 3.2 Plasmid transduced into monocytes………………………………...………17 3.3 Monocytes expressing Luc reporter gene……………………………...……19 3.4 Constructing template vector for Luc reporter gene assay system in monocytes…………………………………………………………………...19 Chapter 4 Discussion 4.1 pBabe backboned vector failed to work in THP-1 cells………………….…21 4.2 NF-kB signaling pathway activated by TNFa and INFr can be detected in our reporter gene assay system………………………………………...……22 4.3 Doxycycline induce NF-kB activity in monocytes……………………..…..22 Chapter 5 Figures………………………………………………………………..……24 Chapter 6 Tables…………………………………………………………………...…32 Chapter 7 References…………………………………………………………………33 Chapter 8 Appendix………………………..…………………………………………37 | |
| dc.language.iso | zh-TW | |
| dc.subject | NF-kB | zh_TW |
| dc.subject | 報導基因 | zh_TW |
| dc.subject | 單核球細胞 | zh_TW |
| dc.subject | THP-1 | zh_TW |
| dc.subject | 螢光素酶 | zh_TW |
| dc.subject | monocyte | en |
| dc.subject | NF-kB | en |
| dc.subject | luciferase | en |
| dc.subject | THP-1 | en |
| dc.subject | reporter gene | en |
| dc.title | 單核球細胞報導基因檢測系統的建立與應用 | zh_TW |
| dc.title | Establishment and Application of a Reporter Gene Assay System in Monocytes | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹世鵬(Hsien-Ho Lin),蘇剛毅(HSIU-HSI CHEN) | |
| dc.subject.keyword | 報導基因,單核球細胞,THP-1,螢光素酶,NF-kB, | zh_TW |
| dc.subject.keyword | reporter gene,monocyte,THP-1,luciferase,NF-kB, | en |
| dc.relation.page | 39 | |
| dc.identifier.doi | 10.6342/NTU202200291 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-02-11 | |
| 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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| U0001-0502202223070400.pdf 授權僅限NTU校內IP使用（校園外請利用VPN校外連線服務） | 5.46 MB | Adobe PDF |
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