跳到主要內容

臺灣博碩士論文加值系統

(3.235.120.150) 您好!臺灣時間:2021/08/06 03:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳孟寰
研究生(外文):Moon-Huan Chen
論文名稱:在人類細胞中建立正向自我調節迴路以應用至抑制細胞分裂之癌症療法
論文名稱(外文):Synthetic Construction of a Positive Auto-regulatory FeedbackLoop in Human Cells Applied to Differentiated Anti-mitoticCancer Therapy
指導教授:黃筱鈞黃筱鈞引用關係
口試委員:朱家瑩江介宏史有伶楊啟伸
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:74
中文關鍵詞:癌症治療抑制細胞分裂合成生物學正向自我調節回饋迴路雙峰分佈
外文關鍵詞:cancer therapysynthetic biologypositive auto-regulationbimodality
相關次數:
  • 被引用被引用:0
  • 點閱點閱:102
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
對於末期癌症的治療中,化學治療和標靶治療扮演很重要的角色。雖然只針對癌症細胞中特定訊息傳遞路徑中的特定調控因子或是酵素的標靶治療已證實所帶來的副作用較少;但在另一方面,這種攻擊特定目標的專一性卻也會使得原本存在,讓細胞本身對藥物具有抗性的突變更容易被選擇出來,而後反而讓腫瘤細胞得以繼續大量繁衍,導致癌症快速的復發,對於整體存活率的提升有限。至於以抑制細胞分裂為對抗方式的化學治療,則會讓體內其他所有能夠不斷生長分裂的細胞都受到影響,像是骨髓的造血幹細胞,進而造成較多較大的副作用,例如白血球數量的低落,使病人的免疫力下降。因此,我們的研究就是希望在以抑制腫瘤細胞週期進行的治療方式為基礎,但於此同時又能讓骨髓幹細胞存活下來,繼續正常地分裂。我們使用合成生物學的方法,希望建構一個可以在人類細胞中表現的分子迴路,能夠具備此種分辨細胞種類及週期狀態的能力,可以更專一且更有效地殺死癌細胞。當中,我們設計了一個正向自我調節回饋迴路(positive auto-regulatory loop),藉由四環素
(Tetracycline)調控轉錄活化的系統,以螢光蛋白作為播報因子,希望能在人類細胞內,產生一個雙峰分佈(bimodal distribution)。意即讓所有癌細胞在進入細胞分裂時,都能有效開啟基因迴圈,確保其大量表現引起細胞凋亡的蛋白質,而讓癌細胞無法逃過死劫;而反之,則正常細胞的蛋白表現量幾乎為零,因而都可存活下來。最終,希望這樣一個分子迴路的設計,對於未來的癌症基因治療上,能夠加以應用,有所
助益。

Among treatments for the advanced-stage cancers, chemotherapy and targeted therapy are the most common ones. The targeted therapies target a specific enzyme or regulator in a specific signaling pathway in cancer cells, which has been proved to have fewer side effects. But on the other hand, this specificity of targeting has been suggested to be prone to select for preexisting secondary mutations that enable cells resistant to the drug, and acquired resistance would result in rapid cancer recurrence, thus the elevation in overall survival is limited. As for anti-mitotic chemotherapy that blocks cell
mitosis, would hence influence survival of other proliferating cells, such as hematopoietic stem cells in bone marrow, and then brings more and deeper side effects. A low blood cell count, for example, would threaten patients’ lives. Therefore, the aim of our research is to inhibit cell cycle for treatment of cancer, while sparing dividing bone marrow cells. With a “synthetic biology” approach, we would construct a cell cycle-targeted molecular device in human cells that is able to sense cell states and differentiate between cancer and normal cells, delivering more specific and effective killing. In this thesis, we designed a positive auto-regulatory loop through
tetracycline-controlled transcriptional activation system, using fluorescence protein as reporters to visualize device performance, with hope to induce a bimodal distribution by
tuning the circuit response. It means that all cancer cells will initiate this genetic circuit effectively upon entering mitosis, ensuring to express the desired toxic proteins, and go
into apoptosis, whereas all normal cells lack toxic protein expression and are able to survive. Consequently, we hope this design of molecular circuit will be applicable to cancer gene therapy in the future.

謝誌 ........................................................ i
摘要 .........................................................v
Abstract.................................................... vi
Table of Contents.......................................... vii
List of Figures and Tables ...................................x
Chapter.1.Introduction .......................................1
1.1. Human cancer therapy.....................................1
1.2. Synthetic biology approach ..............................2
1.3. The purpose of our research .............................2
1.4. The actual regular units and device .....................3
1.5. The tetracycline-responsive regulatory system ...........5
1.6. The thesis organization..................................5
Chapter.2.Materials and Methods ..............................7
2.1. Recombinant DNA construction ............................7
2.1.1. Bacterial strain and vectors...........................7
2.1.2. DNA primers ...........................................8
2.1.3. Plasmid construction ..................................9
2.1.3.1. Gibson Assembly cloning..............................9
2.1.3.2. Site-directed mutagenesis for deletion..............13
2.1.4. Screening and confirmation for the correct plasmids ..14
2.2. Cells and cell culture..................................15
2.3. Transfection of plasmid DNA into human cells............17
2.3.1. Liposome-mediated transfection........................17
2.3.2. Viral infection ......................................17
2.4. Drug treatments for gene expression induction ..........18
2.5. Immobilization of suspension cells for imaging .........18
2.6. Live-cell imaging ......................................19
2.7. Flow cytometry .........................................19
2.8. Data analysis and statistics............................20
Chapter.3.Results and Discussion ............................21
3.1. HL-60 imaging...........................................21
3.2. Construction of positive auto-regulation and imaging....22
3.3. Construction of positive auto-regulation and analysis with internal transfection control................................24
3.4. Comparison of response between circuits with different promoter strength ...........................................28
3.5. Co-transfection with toxic signal expression ...........31
Chapter.4.Conclusions and Future Works.......................33
4.1. Promotion of TRE promoter regulation efficiency by reducing basal leakiness.....................................33
4.2. Cyclin B1 promoter and micro RNA as circuit sensors ....34
4.3. Lentiviral infection into HL-60 and A549 cells .........34
Chapter.5.References.........................................36
Figures and Tables...........................................39

1. Kris M.G., Natale, R.B., et al. Efficacy of Gefitinib, an Inhibitor of the Epidermal Growth Factor Receptor Tyrosine Kinase, in Symptomatic Patients With Non– Small Cell Lung Cancer: A Randomized Trial. AMA 290, 2149-2158 (2003).
2. Jackson, J.R., Patrick, D.R., Dar, M.M. & Huang, P.S. Targeted anti-mitotic therapies: can we improve on tubulin agents? Nat Rev Cancer 7, 107-117 (2007).
3. Pao, W. & Chmielecki, J. Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer 10, 760-774 (2010).
4. Sprinzak, D. & Elowitz, M.B. Reconstruction of genetic circuits. Nature 438, 443-448 (2005).
5. Eldar, A. & Elowitz, M.B. Functional roles for noise in genetic circuits. Nature 467, 167-173 (2010).
6. Purnick, P.E. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol 10, 410-422 (2009).
7. Khalil, A.S. & Collins, J.J. Synthetic biology: applications come of age. Nat Rev Genet 11, 367-379 (2010).
8. Alon, U. Network motifs: theory and experimental approaches. Nat Rev Genet 8, 450-461 (2007).
9. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).
10. Gossen, M., et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766-1769 (1995).
11. Landgraf, P., et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401-1414 (2007).
12. Oltvai, Z.N., Milliman, C.L. & Korsmeyer, S.J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619 (1993).
13. Xie, Z., Wroblewska, L., et al. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307-1311 (2011).
14. Urlinger, S., Baron, U., et al. Exploring the sequence space for tetracycline dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97, 7963-7968 (2000).
15. Koponen, J.K., Kankkonen, H., et al. Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. Gene Therapy 10, 459–466 (2003).
16. Tang, Y., Orth, J.D., Xie, T. & Mitchison, T.J. Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells. Cancer Lett 310, 15-24 (2011).
17. Szymczak, A.L., Workman, C.J., Wang, Y., et al. Correction of multi-gene deficiency in vivo using a single ''self-cleaving'' 2A peptide–based retroviral vector. Nature Biotechnology 22, 589-594 (2004).
18. Kim, J.H., Lee, S.R., Li, L.H., et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011).
19. Tsz-Leung To & Narendra Maheshri. Noise can induce bimodality in positive transcriptional feedback loops without bistability. Science 327, 1142 (2010).
20. Mohammadi, S.A., O’Malley, M., et al. Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness. J Gene Med 6, 817–828
(2004).
21. Loew, R., Heinz, N., et al. Improved Tet-responsive promoters with minimized background expression. BMC Biotechnology 10, 81 (2010).

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
無相關期刊
 
無相關點閱論文