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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
dc.contributor.author | Ling-Yea Yu | en |
dc.contributor.author | 尤鈴雅 | zh_TW |
dc.date.accessioned | 2021-06-17T02:29:38Z | - |
dc.date.available | 2022-08-23 | |
dc.date.copyright | 2017-08-23 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68664 | - |
dc.description.abstract | 細胞遷移(Cell migration)對於許多生物途徑而言十分重要,例如影響胚胎形成、組織再生與癌症轉移。近期許多研究出許多影響細胞遷移的基因,不過這些基因如何交互作用而影響細胞的移動卻仍是未知的。為了解決這個問題,我們做了一個雙擊模式的細胞遷移篩選 (“two-hit” cell migration screen),利用shRNA與小分子抑制劑,看目標基因與小分子藥物對細胞遷移的交互作用。在多數的候選基因中,我們對於STK40這個潛在的serine/threonine激酶十分感興趣。之前的研究指出,減少STK40的量會增加細胞移動速度。然而,我們的雙擊篩選卻發現,同時減少STK40與利用PD98059 這個MEK抑制劑會導致速率驟減。因此,我們推測STK40會與MAPK路徑有交互作用,藉此調節細胞遷移。
為了驗證我們的假說,我們在上皮與內皮細胞中做了許多細胞遷移實驗,分析了不同的細胞遷移相關參數,探討STK40調節細胞遷移的情形。我們發現STK40會增加SAS的細胞移動速度(speed)與細胞間的協調性(coordination),而增加HUVEC的細胞移動速度。我們另外也做了免疫螢光染色實驗,改變STK40的量檢視E-cadherin與phalloidin量的變化,希望能解釋STK40改善細胞間的協調性。未來研究會探討STK40 實際上如何與MAPK調節細胞遷移,希望能找出STK40調節細胞遷移的機轉。 | zh_TW |
dc.description.abstract | Cell migration is important for embryonic development, tissue regeneration and cancer metastasis. Recent research identified cell migration-related genes, but how they crosstalk with each other remain unclear. To resolve this mystery, we conducted a “two-hit” cell migration screen using short hairpin RNA (shRNA) and small molecules inhibitors. Among our candidates, we are particularly interested in STK40, a putative serine/threonine kinase. Previous literature reports showed that STK40 knockdown increased cell motility. However, our two-hit screen revealed STK40 knockdown plus PD98059 (MEK inhibitor) resulted in a dramatic suppression of cell motility. We therefore hypothesize that STK40 interacts with MAPK signaling to regulate cell migration.
To validate this hypothesis, we first conducted various cell migration assays by analyzing different parameters to investigate how STK40 regulates cell migration in epithelial and endothelial cells. We discovered that STK40 improved speed in both cell lines and improved coordination in SAS. Our immunofluorescent assays also showed that altering STK40 level altered E-cadherin and phalloidin, which may explain STK40 improvement in coordination. Further exploration will aim at how STK40 and MAPK regulate cell migration, hoping to disclose the mechanism of the regulation of STK40 in cell migration. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:29:38Z (GMT). No. of bitstreams: 1 ntu-106-R04443010-1.pdf: 5558504 bytes, checksum: 3608bf4ff8226e171f5001f301309883 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................... i 中文摘要 ..................................................................................................................................... iii ABSTRACT ................................................................................................................................ iv LIST OF FIGURES .................................................................................................................... viii LIST OF TABLES ........................................................................................................................ x LIST OF ABBREVIATIONS ...................................................................................................... xi Chapter 1 Introduction ............................................................................................................. 1 1.1 Cell Migration and Cancer Development ...................................................................... 1 1.2 Genes Affecting Cell Migration Identified via Screens ................................................. 2 1.3 “Two-hit” wound healing assay: finding crosstalk between cell-migration genes ........ 3 1.4 Serine/Threonine Kinase (STK40) ................................................................................ 7 1.5 Serine/Threonine Kinase interacts with Mitogen-Activated Protein Kinase (MAPK) .. 8 1.6 Aim: Find out how STK40 regulates cell migration, potentially through interaction with MAPK .................................................................................................................................. 12 Chapter 2 Materials and Methods .......................................................................................... 14 2.1 Cell Lines and Cell Culture ......................................................................................... 14 2.2 Lentivirus-based Silencing by shRNA ........................................................................ 15 2.3 Lentivirus-based Overexpression Constructs .............................................................. 15 2.4 Lentivirus Packaging ................................................................................................... 18 2.5 Titer Calculation .......................................................................................................... 19 2.6 Transduction of Lentiviruses ....................................................................................... 22 2.7 Quantitative Real-Time PCR ....................................................................................... 23 2.8 Western Blot ................................................................................................................ 24 2.9 Cell Migration Assay ................................................................................................... 26 2.10 Immunofluorescent Assay (IFA) ................................................................................. 29 2.11 Statistical Analysis ....................................................................................................... 30 Chapter 3 Results ................................................................................................................... 31 3.1 STK40 is expressed in SAS and HUVEC ................................................................... 31 3.2 Knockdown and overexpression of STK40 in SAS and HUVEC ............................... 32 3.3 STK40 can be knocked-down and overexpressed in SAS ........................................... 32 3.4 STK40 can be successfully overexpressed and but cannot be knocked-down in HUVEC ................................................................................................................................ 34 3.5 Utilizing cell migration assays to decipher how STK40 regulates cell migration ....... 35 3.6 Cell Migration assay of STK40 in SAS ....................................................................... 37 3.7 Cell migration assay of STK40 in HUVEC ................................................................. 39 3.8 STK40 knocked-down SAS cells show increased level of cortical actin and E-cadherin, while overexpressed cells reduce actin and E-cadherin ........................................................ 40 3.9 In search of interaction of STK40 and MAPK: Evaluating MEK inhibitors in Western blot and cell migration .......................................................................................................... 43 Chapter 4 Discussion ............................................................................................................. 46 4.1 STK40 regulation on cell migration may not be simply due to alteration in speed but due to its effect on cell-cell coordination .............................................................................. 46 4.2 Examining all parameters to confirm the principal parameter controlled by STK40 .. 48 4.3 Random migration results implied that STK40 improved cell migration .................... 49 4.4 Wound healing migration aids us in finding the main parameter controlled by STK40 .................................................................................................................................. 50 4.5 Migration assays pointed out that coordination may be the key parameter of STK40 regulation on cell migration .................................................................................................. 52 4.6 Issues on cell density on our migration assays ............................................................ 53 4.7 Issues on transduction efficiency ................................................................................. 55 4.8 Staining phalloidin and E-cadherin in SAS in search of mechanism .......................... 57 4.9 Establishing the machinery of STK40 in epithelial and endothelial cell movement ... 60 4.10 Connecting STK40 to MAPK by studying migration by MEK inhibition .................. 61 4.11 Conclusion ................................................................................................................... 62 4.12 Future direction ............................................................................................................ 63 Chapter 5 Figures ................................................................................................................... 65 Chapter 6 Tables .................................................................................................................... 93 MATLAB SCRIPTS ................................................................................................................... 98 REFERENCE ............................................................................................................................ 123 | |
dc.language.iso | en | |
dc.title | 絲氨酸/蘇氨酸蛋白激酶40與細胞遷移:與絲裂原活化蛋白激酶(MAPK)的交互作用 | zh_TW |
dc.title | Serine Threonine Kinase-40 (STK40) and Cell Migration: Interaction with Mitogen-Activated Protein Kinase (MAPK) | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳青周(Ching-Chow Chen),林耿慧(Keng-Hui Lin) | |
dc.subject.keyword | STK40,MAPK,PD98059,SAS,HUVEC,細胞協調性,細胞遷移, | zh_TW |
dc.subject.keyword | STK40,MAPK,PD98059,SAS,HUVEC,coordination,cell migration, | en |
dc.relation.page | 124 | |
dc.identifier.doi | 10.6342/NTU201703311 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥理學研究所 | zh_TW |
顯示於系所單位: | 藥理學科所 |
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