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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃筱鈞(Hsiao-Chun Huang) | |
| dc.contributor.author | Pei-Yu Chung | en |
| dc.contributor.author | 鍾佩妤 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:30:37Z | - |
| dc.date.available | 2023-08-16 | |
| dc.date.copyright | 2018-08-16 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70542 | - |
| dc.description.abstract | 微型核醣核酸(microRNA)是一種在轉錄後調節基因表現的非編碼核糖核酸，研究指出miR-142 微型核醣核酸在血液相關細胞株中大量表現。在本研究中，我們利用合成基因迴路建立誘導式細胞凋亡路徑，此迴路包含兩個感應元件：細胞週期素 B1 啟動子(cyclinB1 promoter)及 miR-142 微型核醣核酸結合位點(microRNA binding site，MBS)，與一個執行元件：Bcl-2相關X蛋白(hBax protein)。hBax之功能為促進細胞色素c (cytochrome C)釋放進而活化凋亡蛋白酶(caspase)，引發細胞凋亡。此基因迴路經邏輯匣判斷後會在非血液相關細胞株中引發細胞凋亡，而在血液相關細胞株中因有微型核醣核酸結合至微型核醣核酸結合位點，引發核糖核酸干擾(RNA interference, RNAi)，並不會發生細胞凋亡。經實驗證實miR-142-5p比起miR-142-3p在血液細胞株中有更專一之抑制效果，我們藉此以miR-142-5p建立完整的合成基因迴路，並在非血液細胞株中引發至少30%之細胞凋亡。本研究藉由合成生物學跨領域之應用，建立體外實驗中能標定並殺死非血液腫瘤細胞之合成基因迴路，也為標靶基因治療提供了基礎概念驗證及具潛力之新方向。 | zh_TW |
| dc.description.abstract | MicroRNAs are a class of small non-coding RNAs that regulate the gene-silencing process at the post-transcriptional level. Evidence indicates that miR-142 is highly expressed in hematopoietic cell lines. This study employed a synthetic biological circuit that can trigger apoptosis after doxycycline induction. The device contains two sensors: one is an inducible cyclin B1 promoter-driven Tet-On 3G system, and the other is a synthetic miR-142 microRNA binding site (miR-142 MBS) added to the 3'-untranslated region (3'-UTR) of the device’s output signal, human BAX protein (hBax). hBax is a member of the Bcl-2 gene family, which functions as an apoptotic activator of cytochrome C release and caspase activation. The circuit, comprising an biological AND gate, simultaneously senses the initiation of cell division and cell identity, and then triggers the expression of the toxic signal that specifically kills non-hematopoietic cells. The endogenous miR-142 in hematopoietic cells will bind to the mRNA of apoptotic genes of the circuit, and thus prevent apoptosis in the hematopoietic cells. By contrast, activation of the hBax gene leads to an apoptotic cascade in non-hematopoietic cells. Furthermore, our study reveals that miR-142-5p has higher repression efficiency in hematopoietic cancer cell lines (HL-60, Jurkat and CCRF-CEM) than miR-142-3p does. We therefore established our device using miR-142-5p. We also demonstrate that there are at least 30% of cells result in apoptosis in non-hematopoietic cancer cell lines (HeLa, HCT116 and U2OS). We are currently assembling the two sensors and the effector to realize the synthetic circuit that can differentially target non-hematopoietic cancer cell lines. This study provides a basis proof-of-concept for targeted gene therapy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:30:37Z (GMT). No. of bitstreams: 1 ntu-107-R05b43035-1.pdf: 8218439 bytes, checksum: 316ae3a25669fbed2d0eb8534e70fa36 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 I
致謝 II 摘要 IV ABSTRACT V CONTENT VI FIGURE LIST VIII CHAPTER 1 INTRODUCTION 1 1.1 Applications of Synthetic Biology and microRNA Therapeutics 1 1.2 Conception of the Circuit Design 2 1.3 Improvements of Low Transfection Efficiency in Hematopoietic Cell Lines and Low Death Rate in A549 Cell Line 5 1.4 Project Purpose 6 CHAPTER 2 MATERIALS AND METHODS 8 2.1 Recombinant DNA Construction 8 2.1.1 Bacterial Strain and Vectors 8 2.1.2 Oligonucleotide Sequences for Construction 9 2.1.3 Reagents and Enzymes 11 2.2 Cell Lines and Cell Culture 12 2.3 Delivery of Plasmid DNA into Mammalian Cells 13 2.3.1 Liposome-mediated Transfection 14 2.3.2 Electroporation 14 2.4 Drugs Treatment 15 2.5 Fluorescence Microscopy and Time-lapse Imaging 15 2.6 Flow Cytometry Analysis and DAPI Staining 16 2.7 MTS Assay 16 2.8 RNA Extraction and RT-qPCR 17 2.9 Generation of Stable Cell Lines 17 3.0 Data Analysis and Statistics 18 CHAPTER 3 RESULTS AND DISCUSSION 19 3.1 Analysis of MicroRNA-142 Expression in Various Cell Lines 19 3.1.1 Measurement of Endogenous microRNA-142 19 3.1.2 Measurement of MicroRNA Activities in Different Cell Lines 19 3.1.3 Functional Assay of 4X MBS by Overexpressing pre-miR-142 Construct 20 3.2 The Classifier Circuit Design and Killing Experiments in Different Cell Lines 21 3.2.1 Logic Circuit Design and Regulatory Units in the Circuit 21 3.2.2 Consecutive Apoptotic Gene Testing 22 3.2.3 hBax Circuit Performance Analysis 22 3.3 Improvements for Low Transfection Efficiency 24 3.3.1 Generation of Stable Cell Lines by Flow Sorting (FACS) 24 3.3.2 P2A Peptide Function Testing 24 3.4 Combination of Apoptotic Genes Analysis 25 CHAPTER 4 CONCLUSION AND FUTURE WORK 27 FIGURES 29 REFERENCE 54 APPENDIX: GENE MAP 57 | |
| dc.language.iso | en | |
| dc.subject | 癌症 | zh_TW |
| dc.subject | 基因治療 | zh_TW |
| dc.subject | 邏輯匣 | zh_TW |
| dc.subject | 合成生物學 | zh_TW |
| dc.subject | 微型核醣核酸 | zh_TW |
| dc.subject | 核糖核酸干擾 | zh_TW |
| dc.subject | logic gate | en |
| dc.subject | synthetic biology | en |
| dc.subject | microRNA | en |
| dc.subject | gene therapy | en |
| dc.subject | RNA interfering | en |
| dc.title | 以微小核糖核酸表現差異調控非血液癌細胞差別性死亡之生物合成迴路 | zh_TW |
| dc.title | A MicroRNA-based Apoptotic Circuit for Differential Killing of Non-Hematopoietic Cancer Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 江運金(YUN-JIN JIANG),吳?承(HSUAN-CHEN WU) | |
| dc.subject.keyword | 合成生物學,微型核醣核酸,基因治療,邏輯匣,核糖核酸干擾,癌症, | zh_TW |
| dc.subject.keyword | synthetic biology,microRNA,gene therapy,logic gate,RNA interfering, | en |
| dc.relation.page | 67 | |
| dc.identifier.doi | 10.6342/NTU201803124 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-13 | |
| 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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