請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27135
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 于宏燦,李仲良 | |
dc.contributor.author | Yi-Hsuan Chang | en |
dc.contributor.author | 張亦璇 | zh_TW |
dc.date.accessioned | 2021-06-12T17:56:10Z | - |
dc.date.available | 2010-02-18 | |
dc.date.copyright | 2008-02-18 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-01-31 | |
dc.identifier.citation | Alam, J. and J. L. Cook (1990). 'Reporter genes: application to the study of mammalian gene transcription.' Anal Biochem 188(2): 245-54.
Anastasia, L., M. Sampaolesi, et al. (2006). 'Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle.' Cell Death Differ 13(12): 2042-51. Anders, C. K., C. R. Acharya, et al. (2008). 'Age-specific differences in oncogenic pathway deregulation seen in human breast tumors.' PLoS ONE 3(1): e1373. Bhat, R., Y. Xue, et al. (2003). 'Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418.' J Biol Chem 278(46): 45937-45. Chen, S., Q. Zhang, et al. (2004). 'Dedifferentiation of lineage-committed cells by a small molecule.' J Am Chem Soc 126(2): 410-1. Cockrell, A. S. and T. Kafri (2007). 'Gene delivery by lentivirus vectors.' Mol Biotechnol 36(3): 184-204. de Wet, J. R., K. V. Wood, et al. (1987). 'Firefly luciferase gene: structure and expression in mammalian cells.' Mol Cell Biol 7(2): 725-37. Ding, S., T. Y. Wu, et al. (2003). 'Synthetic small molecules that control stem cell fate.' Proc Natl Acad Sci U S A 100(13): 7632-7. Estella, C., D. J. McKay, et al. (2008). 'Molecular Integration of Wingless, Decapentaplegic, and Autoregulatory Inputs into Distalless during Drosophila Leg Development.' Dev Cell 14(1): 86-96. He, B. and D. M. Jablons (2006). 'Wnt signaling in stem cells and lung cancer.' Ernst Schering Found Symp Proc 5: 27-58. He, T. C., A. B. Sparks, et al. (1998). 'Identification of c-MYC as a target of the APC pathway.' Science 281(5382): 1509-12. Higaki, J. N., S. Chakravarty, et al. (1999). 'A combinatorial approach to the identification of dipeptide aldehyde inhibitors of beta-amyloid production.' J Med Chem 42(19): 3889-98. Janssens, N., M. Janicot, et al. (2006). 'The Wnt-dependent signaling pathways as target in oncology drug discovery.' Invest New Drugs 24(4): 263-80. Jing, P., Q. Jin, et al. (2008). 'GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis.' J Neurochem 104(2): 409-19. Katoh, M. and M. Katoh (2007). 'WNT signaling pathway and stem cell signaling network.' Clin Cancer Res 13(14): 4042-5. Kim, J., X. Zhang, et al. (2006). 'Suppression of Wnt signaling by the green tea compound (-)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells. Requirement of the transcriptional repressor HBP1.' J Biol Chem 281(16): 10865-75. Kohn, A. D. and R. T. Moon (2005). 'Wnt and calcium signaling: beta-catenin-independent pathways.' Cell Calcium 38(3-4): 439-46. Kunick, C., K. Lauenroth, et al. (2004). '1-Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3 beta.' Bioorg Med Chem Lett 14(2): 413-6. Leclerc, S., M. Garnier, et al. (2001). 'Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?' J Biol Chem 276(1): 251-60. Liao, Y. F., B. J. Wang, et al. (2007). 'Unnatural amino acid-substituted (hydroxyethyl)urea peptidomimetics inhibit gamma-secretase and promote the neuronal differentiation of neuroblastoma cells.' Mol Pharmacol 71(2): 588-601. Lin, S. Y., W. Xia, et al. (2000). 'Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression.' Proc Natl Acad Sci U S A 97(8): 4262-6. Lo Celso, C., D. M. Prowse, et al. (2004). 'Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours.' Development 131(8): 1787-99. Longo, K. A., J. A. Kennell, et al. (2002). 'Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors.' J Biol Chem 277(41): 38239-44. Meijer, L., A. L. Skaltsounis, et al. (2003). 'GSK-3-selective inhibitors derived from Tyrian purple indirubins.' Chem Biol 10(12): 1255-66. Miyabayashi, T., J. L. Teo, et al. (2007). 'Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency.' Proc Natl Acad Sci U S A 104(13): 5668-73. Moon, R. T., A. D. Kohn, et al. (2004). 'WNT and beta-catenin signalling: diseases and therapies.' Nat Rev Genet 5(9): 691-701. Munemitsu, S., I. Albert, et al. (1996). 'Deletion of an amino-terminal sequence beta-catenin in vivo and promotes hyperphosporylation of the adenomatous polyposis coli tumor suppressor protein.' Mol Cell Biol 16(8): 4088-94. Niemann, S., C. Zhao, et al. (2004). 'Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family.' Am J Hum Genet 74(3): 558-63. Nusse, R. and H. E. Varmus (1982). 'Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.' Cell 31(1): 99-109. O'Neill, D. J., L. Shen, et al. (2004). 'Design, synthesis, and biological evaluation of novel 7-azaindolyl-heteroaryl-maleimides as potent and selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors.' Bioorg Med Chem 12(12): 3167-85. Ow, D. W., D. E. W. JR, et al. (1986). 'Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants.' Science 234(4778): 856-859. Park, C. H., J. Y. Chang, et al. (2005). 'Quercetin, a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells.' Biochem Biophys Res Commun 328(1): 227-34. Polakis, P. (2000). 'Wnt signaling and cancer.' Genes Dev 14(15): 1837-51. Reya, T. and H. Clevers (2005). 'Wnt signalling in stem cells and cancer.' Nature 434(7035): 843-50. Sato, N., L. Meijer, et al. (2004). 'Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.' Nat Med 10(1): 55-63. Schultz, C., A. Link, et al. (1999). 'Paullones, a series of cyclin-dependent kinase inhibitors: synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity.' J Med Chem 42(15): 2909-19. Segditsas, S. and I. Tomlinson (2006). 'Colorectal cancer and genetic alterations in the Wnt pathway.' Oncogene 25(57): 7531-7. Shtutman, M., J. Zhurinsky, et al. (1999). 'The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway.' Proc Natl Acad Sci U S A 96(10): 5522-7. Singh, R., J. N. Artaza, et al. (2006). 'Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors.' Endocrinology 147(1): 141-54. Somjen, D., K. Tordjman, et al. (2007). 'Lipoxygenase metabolites are mediators of PTH-dependent human osteoblast growth.' Bone. Strochkova, L. S. (1978). '[Effect of inhibitors of RNA and protein synthesis on the course of mitosis in a synchronized culture of Chinese hamster cells].' Biull Eksp Biol Med 85(6): 739-43. Sugimoto, Y., S. Nakamura, et al. (2008). 'HOXA10 expression induced by Abl kinase inhibitors enhanced apoptosis through PI3K pathway in CML cells.' Leuk Res. Teo, R., F. Mohrlen, et al. (2006). 'An evolutionary conserved role of Wnt signaling in stem cell fate decision.' Dev Biol 289(1): 91-9. Thangapazham, R. L., N. Passi, et al. (2007). 'Green Tea Polyphenol and Epigallocatechin Gallate Induce Apoptosis and Inhibit Invasion in Human Breast Cancer Cells.' Cancer Biol Ther 6(12). Veeman, M. T., D. C. Slusarski, et al. (2003). 'Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements.' Curr Biol 13(8): 680-5. Willert, K., J. D. Brown, et al. (2003). 'Wnt proteins are lipid-modified and can act as stem cell growth factors.' Nature 423(6938): 448-52. Willert, K. and K. A. Jones (2006). 'Wnt signaling: is the party in the nucleus?' Genes Dev 20(11): 1394-404. Wodarz, A. and R. Nusse (1998). 'Mechanisms of Wnt signaling in development.' Annu Rev Cell Dev Biol 14: 59-88. Wu, X., S. Ding, et al. (2004). 'Small molecules that induce cardiomyogenesis in embryonic stem cells.' J Am Chem Soc 126(6): 1590-1. Wu, X., J. Walker, et al. (2004). 'Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway.' Chem Biol 11(9): 1229-38. Ysebaert, L., G. Chicanne, et al. (2006). 'Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis.' Leukemia 20(7): 1211-6. Zeng, G., U. Apte, et al. (2007). 'siRNA-mediated beta-catenin knockdown in human hepatoma cells results in decreased growth and survival.' Neoplasia 9(11): 951-9. Zhu, A. J. and F. M. Watt (1999). 'beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion.' Development 126(10): 2285-98. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27135 | - |
dc.description.abstract | Wnt signaling pathway在多細胞動物中具有高度保留性。此訊息傳導途徑可於發育時期調節身體結構(body patterning),並且終身都具有決定幹細胞命運的能力。根據其對於特定組織幹細胞的影響力,在這些特定組織中癌症也隨著Wnt 訊息傳導途徑的異常活化而發生。此一關係顯示了尋找幹細胞及癌細胞之外來Wnt 訊息傳導途徑調節物的重要性及進一步運用於再生醫學及癌症治療的潛力。
此研究的目的在於建立一個以細胞為平台的報導系統,此系統可做為一個強而有力並具對於Wnt 訊息傳導途徑專一性的指標,之後並依此系統篩選新的小分子調節物。在比較過兩種小鼠的纖維母細胞株後,3T3-L1細胞被選為建立Wnt 訊息傳導途徑報導系統的模式細胞株。我們所建立帶有穩定結合Wnt 訊息傳導途徑報導質體Super(8X)TopFlash的3T3-L1 B2細胞株,可以表現對於外來Wnt-3a分子在細胞數、作用時間、劑量上相關的的專一反應。在利用九個GSK3抑制劑及六個與Wnt 訊息傳導途徑不相關的已知化學小分子的預先篩選上,有四個GSK3抑制劑可以誘導冷光酶(luciferase)活性的產生,與Wnt 訊息傳導途徑不相關的化學小分子則皆不具有此能力,此結果確認了此系統在篩選Wnt/beta-catenin 訊息傳導途徑小分子活化物的應用性。當處理以連續稀釋的已知Wnt 訊息傳導途徑抑制物,包括槲黃素(Quercetin)及兒茶素益多酚(EGCG)後,由50% L Wnt-3a條件培養液(conditioned medium)所誘導產生的冷光酶(luciferase)活性出現了具劑量相關的被抑制現象,說明了B2細胞株可應用在篩選Wnt/beta-catenin 訊息傳導途徑小分子抑制物的能力。在篩選過兩個系列的小分子,一個系列為合成的gamma-secretase 可能抑制物,另一為植物萃取物及其衍生物後,我們找到了兩個Wnt/beta-catenin 訊息傳導途徑的活化物及十五個抑制物。這兩個活化物名為SY202及SY203,十五個抑制物包括了九個合成的化學物及六個植物萃取物,九個合成的化學物分別名為Jia138、SY15、SY57、SY85、SY173、SY186、SY188、SY202、SY203,六個植物萃取物則包含CCL GS-SE、CCL IC-F、CCL kts、CCL ktf、CCL CD-X、CCL BB。在進一步的半定量(titration)實驗中,四個合成化學物包括SY186、SY188、SY202、SY203同時顯示了Wnt/beta-catenin 訊息傳導途徑活化物及抑制物的活性。根據這些合成化合物在之前的實驗中所表現的gamma-secretase抑制物活性,這些化合物也許可以做為一個探針,幫助我們進一步了解Wnt與Notch兩個訊息傳導途徑間的聯繫情形。經由後續對於這些新Wnt/beta-catenin 訊息傳導途徑調節物的確認及功能性分析,我們期待在不久的將來,這些新調節物能在幹細胞及癌症研究上有所貢獻。 | zh_TW |
dc.description.abstract | Wnt signaling pathway is highly conserved in metazoan. This pathway regulates body patterning during development and determinates stem cell fate throughout lifetime. With its influence in the stem cell from specific tissue, cancer ensues upon dysregulated activation of the Wnt pathway. This connection reveals the importance of searching extrinsic modulators of the Wnt signaling pathway in stem cells and cancer cells with potential applications in regenerative medicine and cancer therapy.
The aim of this research is to establish a cell-based reporter system which can be used as a robust specific indicator for the canonical Wnt signaling pathway and to screen for small novel modulators. After comparing two mouse fibroblastoid cell lines, 3T3-L1 was chosen as a model cell line to establish the Wnt/beta-catenin signaling reporter system. The established 3T3-L1 B2 cell clone, which harbored a stably integrated Super (8X) TopFlash Wnt signaling reporter plasmid, exhibits a specific response to exogenous Wnt3A molecule in a cell number-, time- and dose-dependent manner. With the prescreening of known modulators, including 9 GSK3 inhibitors and 6 irrelevant small chemicals, 4 GSK3 inhibitors but no irrelevant small chemicals showed positive response, confirmed the practicable ability of this system in screening novel Wnt/beta-catenin signaling activators. When treated with serious diluted well-documented Wnt signaling inhibitors, Quercetin and EGCG, a dose-dependent inhibitory curve was revealed in the present of 50% L Wnt3a CM, illustrating the B2 cell clone can be applied in screening novel Wnt/beta-catenin signaling inhibitors. There are two activators and fourteen inhibitors of Wnt/beta-catenin signaling pathway found by the established system from the screening of two libraries including one synthesized compounds and another plant extracts. Four compounds showed a biphasic activity of Wnt signaling in the further titration. With the gamma-secretase inhibitor activity revealed in previous data, these compounds might be probes for investigation the crosstalk of Wnt/Notch signaling in the future. By further identification and functional assay of the new candidate modulators, we expect these new modulators could be used in stem cell and cancer research in the near future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T17:56:10Z (GMT). No. of bitstreams: 1 ntu-97-R94b41028-1.pdf: 722114 bytes, checksum: a7a9bfd93755d8f9cbfd46b1f6f04213 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | Table of Contents
Abstract in English i Abstract in Chinese 中文摘要 iii Table of Contents v List of Tables viii List of Figures ix 1. Introduction 1 1.1 Wnt Signaling Pathway 1 1.1.1 The Canonical Wnt Pathway 1 1.1.2 Wnt Pathway in Development 3 1.1.3 Wnt Pathway in Cancer 4 1.1.4 Wnt Pathway in Stem Cells 5 1.1.4.1 HSC 6 1.1.4.2 ESC 7 1.1.5 Activation of Wnt/beta-catenin Pathway 8 1.1.5.1 L-Wnt3a Condition Medium 8 1.1.5.2 Recombinant Proteins 8 1.1.5.3 Small Molecules 9 1.1.5.4 Gene Transduction 9 1.1.6 Super8XTOPFlash(M50)Vector and Lucuferase Assay 10 1.2. Small Modulators of Wnt/beta-catenin Pathway 11 1.2.1 Activators 12 1.2.1.1 BIO 12 1.2.1.2 TWS119 13 1.2.1.3 Indirubin 13 1.2.2 Suppressors 13 1.2.2.1 EGCG 13 1.2.2.2 Quercetin 14 1.3 Thesis objective and overview 14 2. Materials & Methods 16 2.1 GF/cytokine and Chemicals 16 2.2 The two libraries were screened for Wnt/beta-catenin signaling modulators 17 2.3 Cell culture 18 2.4 L Wnt-3a condition medium 19 2.5 Luciferase assay 20 2.6 beta-galactosidase assay 22 2.7 Normalized luciferase activity 23 2.8 DNA Transfection 23 2.9 MTS assay 24 2.10 Viral production and Infection of target cells 24 3. Results 26 3.1 Establishment of The Cell-Based Screening System 26 3.1.1 Screening of cell lines for the reporter system of Wnt/beta-catenin signaling 26 3.1.2 Establishment of 3T3-L1 cell clones stably expressing the Super (8X) TopFlash, M50 27 3.1.3 Characterization of the B2 cell clone 28 3.1.3.1 Luciferase activation of B2 clone is specific to exogenous stimulation of rmWnt3a molecule 28 3.1.3.2 Dose-dependent manner of the recombinant mouse Wnt3a protein and L Wnt-3a condition medium 29 3.1.3.3 Optimization of Wnt3a assay in the 96 well plate formation 30 3.1.3.4 The use of B2 clone in the GSK3 inhibitors, as well as Wnt signaling activators. 31 3.1.3.5 The use of B2 clone for the screening of Wnt signaling inhibitors. 33 3.2 Screening of The Novel Modulators of The Wnt/beta-catenin Signaling Pathway34 3.2.1 Activator 35 3.2.2 Inhibitor 36 3.2.3 Further identification and titration of the new candidate modulators 37 3.3 Establishment of clone B2 cell stably expressing the regulatable beta-catenin 38 4. Discussion 40 5. Reference 48 | |
dc.language.iso | en | |
dc.title | Canonical Wnt Signaling Pathway 小分子化學調節物細胞篩選平臺之建立 | zh_TW |
dc.title | Development of a cell-based reporter system for the screening of small chemical modulator(s) of the canonical Wnt signaling pathway | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 廖永豐,沈家寧 | |
dc.subject.keyword | Wnt 訊息傳導途徑,細胞篩選平台:小分子調節物, | zh_TW |
dc.subject.keyword | Wnt signaling pathway,cell-based screening system,small novel modulator(s), | en |
dc.relation.page | 79 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-02-01 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 動物學研究所 | zh_TW |
顯示於系所單位: | 動物學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-97-1.pdf 目前未授權公開取用 | 705.19 kB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。