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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77182完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲 | |
| dc.contributor.author | Shih-En Chou | en |
| dc.contributor.author | 周詩恩 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:49:43Z | - |
| dc.date.available | 2021-07-10T21:49:43Z | - |
| dc.date.copyright | 2019-08-29 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77182 | - |
| dc.description.abstract | 正常的人體細胞因為端粒縮短所以不能無限分裂，這種現象稱為細胞老化。端粒縮短會誘導 DNA 損傷反應以阻止細胞週期停滯。DNA 損傷途徑主要由一系列蛋白質的磷酸化和去磷酸化介導。激酶是可以進行多種基質磷酸化的酵素，包括蛋白質和小分子代謝物。為了找出參與在細胞老化中的激酶，我們的實驗室應用基於活性的蛋白質分析 (ABPP)策略來篩選其活性在年輕和老化細胞中發生改變的激酶。在該分析中使用選擇性標記活性激酶的化學探針LCL16001。在本篇研究中，我們首先分析了質譜數據，並選擇了三種激酶，ADK (adenosine kinase)、AK4 (adenylate kinase 4)和 AK5 (adenylate kinase 5)進行進一步分析。這三種激酶參與腺嘌呤生物合成反應，發現它們的活性在老化細胞中升高。使用基於 LCL16001 的 ABPP 方法，我證實了 ADK、AK4 和 AK5 的激酶活性在老化細胞中升高。有趣的是，只有AK4 在老化細胞中顯示出蛋白表現有增加，老化細胞中 ADK 和 AK5 蛋白表現則無。我同時也發現 AK4 的升高不是由於老化細胞中轉錄因子 p53 的激活。為了測試這三種激酶在老化中的作用，我通過腺病毒感染過度表達這些基因，發現它們都沒有誘導細胞的老化。由於激酶活性在細胞中受到嚴格調節，因此實驗可能需要這些基因constitutively-active 突變。有趣的是，通過short hairpin RNAs 降低這三個基因中任一個的表達都會導致正常人成纖維細胞的老化。因此，雖然尚不清楚ADK、 AK4 和 AK5 如何參與衰老，但它們對於防止細胞老化是必要的。 | zh_TW |
| dc.description.abstract | Due to telomere shortening, normal human somatic cells cannot divide infinitely. This phenomenon is called cellular senescence. Telomere shortening induces DNA damage response to arrest cell cycle arrest. DNA damage pathway is mainly mediated by a series of phosphorylation and de-phosphorylation of proteins. Kinases are enzymes that carry out phosphorylation of a variety of substrates including proteins and small molecule metabolites. In order to identify kinases that are involved in cellular senescence, our lab applied activity-based protein profiling (ABPP) strategy to screen for kinases whose activities are altered in proliferating and senescent cells. A chemical probe, LCL16001, that selectively labels active kinases was used in this analysis. In this study, we first analyzed the mass spectrometry data and selected three kinases, ADK (adenosine kinase), AK4 (adenylate kinase 4) and AK5 (adenylate kinase 5) for further analysis. These three kinases are involved in adenine biosynthesis process and their activities were found to be elevated in senescent cells. Using a LCL16001-based activity assay system, I confirmed the kinase activities of ADK, AK4 and AK5 are elevated in senescent cells. Interestingly, only AK4 showed increased protein level in senescent cells. The ADK and AK5 protein levels were not significantly increased in senescent cells. I also found the elevation of AK4 was not due to the activation of transcription factor p53 in senescent cells. To test the role of these three kinases in senescence, I overexpressed these genes by adenovirus infection and found that none of them induced senescence in normal human fibroblasts. Since the kinase activities are tightly regulated in cells, the constitutively-active mutant of these genes might be required for the experiment. Interestingly, knocking down the expression of either one of these three genes by short hairpin RNAs resulted in senescence of normal human fibroblasts. Thus, although it is not clear how ADK, AK4 and AK5 are involved in senescence, they are required to prevent cells from senescence. | en |
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| dc.description.tableofcontents | 目錄 i
表目錄 iii 圖目錄 iv 縮寫檢索表 v 摘要 1 Abstract 2 一、緒論 3 1.1 細胞老化 (Cellular senescence) 3 1.2 激酶 (Kinase) 5 1.3 蛋白質表現量及活性分析 7 1.4 化學活性探針 (Activity-based chemical probe) 8 1.5 研究參與在老化過程的腺嘌呤合成中的激酶 9 二、材料與方法 11 2.1 細胞株培養 (Cell Culture) 11 2.1.1 細胞株來源及培養液 11 2.1.2 細胞株培養 11 2.2 細胞計數 12 2.3 生長曲線 (Population doubling curve) 12 2.4 蛋白質樣品處理及濃度測定 12 2.4.1 樣品處理 12 2.4.2 樣品濃度測定 12 2.5 蛋白質分析蛋白質分析_西方墨點法西方墨點法 (Western blotting analysis) ........................................ 13 2.5.1 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 13 2.5.2 轉漬轉漬 (Transfer) 13 2.5.3 免疫轉漬分析免疫轉漬分析 (Immunoblotting) 13 2.6 化學探針標定反應化學探針標定反應 14 2.6.1 細胞樣品前處理細胞樣品前處理 14 2.6.2 PD-10脫鹽管柱去處未反應脫鹽管柱去處未反應LCL16001探針探針 14 2.6.3 探針標定蛋白質分析探針標定蛋白質分析 14 2.7 RNA萃取與反轉錄聚合酶反應萃取與反轉錄聚合酶反應 (RNA Extraction and Reverse Transcription-PCR) 15 2.7.1 RNA萃取萃取 15 2.7.2 反轉錄反應反轉錄反應 (Reverse transcription, RT) 15 2.7.3 即時定量聚合酶反應即時定量聚合酶反應 (Real-time Polymerase chain reaction) ........................ 15 2.8 Senescence-associated β-galactosidase staining (SA-β-gal staining).......................... 16 2.9 溴化去氧尿苷溴化去氧尿苷 (BrdU)與免疫螢光染色與免疫螢光染色 (Immunofluorescent staining) .......... 17 2.11 活性氧測試活性氧測試 (ROS detection assay) 17 2.12 慢病毒之製備慢病毒之製備 (Lentivirus production)18 2.12.1 質體來源質體來源 18 2.12.2 質體製備質體製備 18 2.12.3 轉染作用轉染作用 (Transfection)與慢病毒生產與慢病毒生產 (Lentivirus production) ................ 18 2.12.4 慢病毒效價測定慢病毒效價測定 (Lentivirus determination) ....................................................... 19 2.13 建構載體建構載體 (Construct)19 2.13.1 聚合酶鏈鎖反應聚合酶鏈鎖反應 (polymerase chain reaction，，PCR) 19 2.13.2 接合反應接合反應 (Ligation) 20 2.13.3 轉殖反應轉殖反應 (Transformation) 20 2.14 腺病毒之置備腺病毒之置備 (Adenovirus production) 20 2.14.1 重重組腺病毒質體組腺病毒質體 (Recombination adenovirus plasmid) ............................................ 20 2.14.2 轉染作用轉染作用 (Transfection)與腺病毒生產與腺病毒生產 (Adenovirus production) ............ 21 2.14.3 腺病毒效價測定腺病毒效價測定 (Adenovirus determination)............................ 21 三、 實驗結果 22 3.1 分析細胞分析細胞IMR90的老化現象的老化現象 22 3.2 使用質譜儀分析使用質譜儀分析LCL16001探針標定之激酶探針標定之激酶 22 3.3 利用質譜儀找到並參與在核苷酸生成過程的激酶利用質譜儀找到並參與在核苷酸生成過程的激酶 24 3.4 利用探針標定驗證質譜儀分析結果利用探針標定驗證質譜儀分析結果 25 3.5 分別過度表現分別過度表現ADK、、AK4、、AK5基因不會使基因不會使IMR90細胞老化細胞老化 ........................ 25 3.6 降低降低ADK基因的表現導致基因的表現導致IMR90細胞走向老化細胞走向老化 26 3.7 降低降低AK4基因的表現導致基因的表現導致IMR90細胞走向老化細胞走向老化 27 3.8 降低降低AK5基因的表現導致基因的表現導致IMR90細胞走向老化細胞走向老化 28 3.9 分別降低分別降低ADK、、AK4及及AK5基因表現基因表現不會使細胞中的氧化壓力上升不會使細胞中的氧化壓力上升 28 3.10 AK4 mRNA及蛋白表現不受降低及蛋白表現不受降低p53基因的表現影響基因的表現影響 29 四、 討論 30 參考文獻35 表目錄 Table 1. shRNAs from National RNAi Core Facility45 Table 2. Biological process of identified kinase by Gene ontology analysis .......................... 46 圖目錄 Figure 1 Senescence phenotype of IMR90 cell line. 47 Figure. 2 Identification probe-labeled kinases in proliferating and senescent IMR90 cells.50 Figure 3 Nucleotide biosynthesis pathway with kinases involved. 51 Figure 4 Schematic presentation activity-based proteomic analysis using chemical probe LCL16001. 52 Figure. 5 Analysis nucleotide biosynthesis kinases in proliferating and senescent IMR90 cells.54 Figure. 6 Overexpression GFP does not make IMR90 cells senescence. .................................... 56 Figure. 7 Overexpression ADK in IMR90 cells can not lead to cellular senescence. 58 Figure. 8 Overexpression AK4 in IMR90 cells can not lead to cellular senescence. .. 60 Figure. 9 Overexpression AK5 in IMR90 cells can not lead to cellular senescence. .. 62 Figure. 10 ADK knockdown induces cellular senescence in IMR90 cells. .............................. 66 Figure. 11 AK4 knockdown induces cellular senescence in IMR90 cells. ................................ 70 Figure. 12 AK5 knockdown induces cellular senescence in IMR90 cells. ................................ 74 Figure. 13 Knockdown ADK, AK4 or AK5 do not increase oxidative stress in IMR90 cells based on ROS. 75 Figure. 14 AK4 expression is not affected by p53 knockdown 76 Appendix 1 Plasmid and competent cell of AdEAsy system. 77 Appendix 2 Structure and labeling mechanism of chemical probe LCL16001. ................ 78 Appendix 3 Mass spectrometry analysis of probe-labeled kinase in young and senescence IMR90 cells. ....... 79 | |
| dc.language.iso | zh-TW | |
| dc.title | 分析腺嘌呤生合成激酶在細胞老化中的角色 | zh_TW |
| dc.title | Analyzing the roles of adenine biosynthesis kinases in cellular senescence | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄,吳青錫 | |
| dc.subject.keyword | 激?,老化,腺嘌呤, | zh_TW |
| dc.subject.keyword | kinases,senescence,adenine, | en |
| dc.relation.page | 80 | |
| dc.identifier.doi | 10.6342/NTU201903930 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-08-19 | |
| 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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