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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲 | zh_TW |
| dc.contributor.advisor | Jing-Jer Ling | en |
| dc.contributor.author | 廖竹旋 | zh_TW |
| dc.contributor.author | Ju-Shiuan Liao | en |
| dc.date.accessioned | 2021-07-10T22:03:06Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-05 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | [1] Hayflick, L., and Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Experimental cell research, 25(3), 585-621.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77461 | - |
| dc.description.abstract | SerpinB2是一種絲胺酸/半胱氨酸蛋白酶抑制劑，廣泛已知它能夠抑制血纖維蛋白溶解酶原的活化過程，進而促進凝血反應。它是藉由與目標蛋白酶催化活性位之間形成共價鍵結的方式，永久性地抑制對方的活性。有趣的是，文獻指出serpinB2是老化的標記蛋白之一，細胞老化的過程serpinB2蛋白表現量也會隨之上升。我們發現在年輕的人類正常纖維母細胞中以腺病毒感染的方式大量表現serpinB2會導致細胞老化；反之，抑制serpinB2的基因表現能夠保護細胞不進入老化的階段。上述結果皆顯示serpinB2在細胞老化的過程中扮演重要的角色。進一步我們也發現serpinB2誘發細胞老化的過程需要TGM2的參與。TGM2是轉麩醯胺酶的一種，廣泛分布存在體內各個組織器官中，它能夠轉移蛋白質上的Glutamine residue的氨基，並與其它蛋白或是多氨化合物之間形成穩定的異肽鍵鍵結，過程中受到鈣離子的濃度所調控。我們發現細胞老化的過程，serpinB2與TGM2之間存在交互作用 (免疫共沉澱的實驗) 並會活化TGM2；同樣地，抑制TGM2的基因表現也能夠保護細胞免於老化的命運。本篇研究中，我發現體外的條件下，serpinB2無法活化TGM2酵素活性，而且它們之間不存在直接的交互作用。因此我們認為細胞老化的條件下應有其他的蛋白幫助serpinB2間接活化TGM2，接著進一步Mass spectrometry分析的結果找出老化過程TGM2特定的目標基質，希望藉此能夠幫助我們進一步探討TGM2參與在serpinB2調控的細胞老化之中所扮演的角色。 | zh_TW |
| dc.description.abstract | SerpinB2 is a serine protease inhibitor that is also known as plasminogen activator inhibitor type 2 (PAI-2). It covalently binds to urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) to inhibit their activities. Interestingly, serpinB2 level is also increased in senescent cells and is considered as a senescence biomarker. Previously, we found elevated protein level of serpinB2 is sufficient to induce normal human fibroblasts into senescence. Moreover, depletion of serpinB2 prevented cells from senescence. These results provide evidence that serpinB2 has a critical role in senescence. We have also found that the serpinB2-mediated cellular senescence is dependent on transglutaminase TGM2. TGM2 is a calcium-dependent transglutaminase, which catalyzes stable isopeptide bond formation between lysine and glutamine residues to crosslink proteins. The transglutaminase activity of TGM2 is increased in senescent cells. Depletion of TGM2 prevents cells from serpinB2-induced senescence. These results support a role of TGM2 in senescence. In this study, I found there is no direct interaction between TGM2 and serpinB2, although the co-immunoprecipitation experiments suggested that serpinB2 interacts with TGM2. I also found that serpinB2 did not greatly affect TGM2 activity, even inhibit it, in contrast to the in vivo finding that serpinB2 activates TGM2 activity. Mass analysis was next conducted to identify senescence-specific TGM2 targets. The identified protein may help resolving the mehanism of how TGM2 is involved in serpinB2-mediated cellular senescence. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T22:03:06Z (GMT). No. of bitstreams: 1 ntu-107-R04442003-1.pdf: 5979167 bytes, checksum: 81a9c9c9f1cda578cf055b2f3f67d8ca (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書 #
誌謝 1 中文摘要 2 Abstract 3 Contents 4 List of Abbreviation Table 7 List of Figures 8 List of Tables 9 Introduction 10 1.1 Cellular Senescence 10 1.2 SerpinB2-induced cellular senescence 12 1.3 Serine Protease Inhibitor, Clade B, Type 2 14 1.4 Transglutaminase 2 in cellular senescence 17 Materials and Method 18 1.1 Cell Lines and Cell Culture 18 1.1.1 細胞株 18 1.1.2 細胞培養液 (Growth Medium) 18 1.1.3 細胞繼代培養 (Subculture of Adherent Cell) 19 1.1.4 細胞計數 (Cell Counting) 19 1.2 Bacterial Strain (Competent Cell) & Bacterial Culture 20 1.2.1 大腸桿菌勝任細胞菌株種類 20 1.2.2 大腸桿菌勝任細胞製備 (Chemically and Electro-competent cell) 20 1.2.3 大腸桿菌勝任細胞轉形作用 (Transformation) 21 1.2.4 細菌培養 22 1.3 Adenovirus Reproduction 22 1.3.1 細胞株 22 1.3.2 腺病毒產量放大 23 1.3.3 腺病毒初步濃縮與純化 23 1.3.4 偵測病毒感染效力 (病毒感染效力) 24 1.3.5 偵測病毒在不同細胞中的感染劑量 (M.O.I) 24 1.4 Lentivirus Production 25 1.4.1 細胞株 25 1.4.2 菌株種類與來源 25 1.4.3 質體純化與限制酶分析 25 1.4.4 轉染 (Transfection) 細胞與收集重組慢病毒 26 1.4.5 偵測病毒相對感染效力 (RIU, Relative Infection Unit) 27 1.4.6 偵測病毒在不同細胞中的感染劑量 (M.O.I) 28 1.5 Western Blot Analysis 28 1.5.1 收集細胞樣品 28 1.5.2 震破細胞取得細胞萃取物 (cell extract) 29 1.5.3 蛋白質濃度測定 (Bradford Protein Assay) 29 1.5.4 聚丙烯醯胺膠體電泳 (SDS PAGE Electrophoresis) 30 1.5.5 轉漬步驟 (Transfer to NC Membrane) 31 1.5.6 免疫轉漬分析 (Immunoblot Analysis) 31 1.6 Protein Purification 32 1.6.1 Plasmid Construction 32 1.6.2 Induction Time Test 33 1.6.3 Protein Purification 33 1.6.4 Coomassie Blue Staining 34 1.7 TGM2 Incorporation Assay 34 1.7.1 Enzyme Reaction 34 1.7.2 Dot Blot Analysis 35 1.7.3 Image Quantification 35 1.8 Mass Spectrometry 35 1.8.1 細胞樣品前處理Pull Down Assay 35 1.8.2 西方墨點法確認蛋白位置 36 1.8.3 上機前樣品前處理 (Gel-assisted digestion for mass spectrometry) 36 Results 38 Discussion 47 References 50 Figure 61 Table 79 | - |
| dc.language.iso | zh_TW | - |
| dc.title | 探討TGM2參與在SerpinB2調控的細胞老化中所扮演的角色 | zh_TW |
| dc.title | The role of TGM2 in serpinB2-mediated cellular senescence | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 鄧述諄;吳青錫 | zh_TW |
| dc.contributor.oralexamcommittee | Shu-Chun Teng;Ching-Shyi Wu | en |
| dc.subject.keyword | serpinB2,細胞老化,TGM2, | zh_TW |
| dc.subject.keyword | serpinB2,cellular senescence,TGM2, | en |
| dc.relation.page | 100 | - |
| dc.identifier.doi | 10.6342/NTU201804027 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-20 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
| 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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