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系統識別號 U0026-0708201515015300
論文名稱(中文) 前胸腺素藉由調控Smad7乙醯化在發炎性腸道疾病中扮演重要角色
論文名稱(英文) Prothymosin α has a critical role in the development of inflammatory bowel disease through regulation of Smad7 acetylation
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 103
學期 2
出版年 104
研究生(中文) 李冠毅
研究生(英文) Kuan-I Lee
學號 S16021133
學位類別 碩士
語文別 中文
論文頁數 53頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-陳炯瑜
口試委員-林季千
口試委員-張孟雅
中文關鍵字 前胸腺素  發炎性腸道疾病  Smad7  乙醯化Smad7 
英文關鍵字 Prothymosin α  Inflammatory bowel disease  Smad7  acetyl-Smad7 
學科別分類
中文摘要 發炎性腸道疾病(inflammatory bowel disease, IBD)是一在大腸和小腸有發炎情形的疾病,主要依據發病部位的不同有克隆氏症及潰瘍性結腸炎兩種類型。研究顯示患有IBD的病人是形成大腸癌的高危險群。前胸腺素(prothymosin α, ProT)是個酸性核蛋白,在細胞增生、轉錄、染色質的重組及凋亡(apoptosis)扮演重要角色。並且在之前實驗室研究指出,在ProT過量表現的轉殖基因小鼠中會自發性的產生肺氣腫以及多囊性腎病。所以本篇研究的就是ProT的過量表現是否參與IBD的發病機制。先分別在臨床IBD病人的Gene Expression Omnibus (GEO) microarray以及硫酸葡聚糖鈉鹽(dextran sulfate sodium, DSS)誘導的形成IBD的小鼠中,均發現ProT有過量表現的現象。在實驗中利用免疫組織化學染色以及觀察小鼠病理切片、病徵大腸長度得知在DSS所誘導的IBD模式下,ProT過量表現的小鼠中發現IBD症狀比對照組嚴重,也觀察到Smad7和Smad7乙醯化也有過量表現的現象,跟先前文獻發現臨床IBD病人的結果一致。而ProT及Smad7基因剔除 (Knockout) 小鼠對DSS所誘發的IBD有較高的耐受性。因此從實驗結果得知ProT在腸道的過量表現會藉由調控Smad7的乙醯化進而產生腸道發炎的症狀。
英文摘要 Inflammatory bowel disease (IBD) is a group of inflammatory condition of the colon and small intestine. Two major types of IBD are ulcerative colitis and Crohn's disease. Chronic inflammation may increase the risk of colon cancer. Prothymosin α (ProT) is an acidic nuclear protein, which is known to play an important role in proliferation, chromatin remodeling, and apoptosis. We have demonstrated previously that ProT transgenic mice exhibit emphysema and polycystic kidney disease (PKD) phenotypes. In this study, we further investigated whether overexpression of ProT is involved in the pathogenesis of IBD. Analyses of microarray data of patients with IBD from the Gene Expression Omnibus (GEO) and our results from dextran sulfate sodium (DSS)-induced colities mice revealed that ProT was overexpressed in the colon of both human and mouse IBD specimens. In the DSS-induced colitis mouse model, ProT transgenic mice expressed higher levels of ProT, Smad7, acetyl-Smad7, and proinflammatory cytokines and had shorter colon length and lower body weight compared to wild-type mice. ProT overexpression increase the acetylation, and activity of Smad7, which antagonises TGF-β signalling. By contract, DSS-induced colitis could be ameliorated by knockdown of ProT expression, which led to Smad7 downregulation in the intestine. In conclusion, our results indicate that excessive expression of ProT in the intestine may play an important role in the regulation of smad7 acetylation in inflammatory conditions during the IBD process.
論文目次 考試合格證明I
英文延深摘要要 II
致謝VIII
縮寫XV
緒論1
1. 發炎性腸道疾病 (Inflammatory bowel disease, IBD)1
1.1 定義與簡介1
1.2 成因2
1.3 診斷與治療2
1.4 IBD動物模式3
2. 前胸腺素 (Prothymosin α, ProT)4
2.1 功能簡介4
2.2 ProT與IBD5
3. Smad7 (Mothers against decapentaplegic homolog 7)5
3.1 功能簡介5
3.2 Smad7與IBD 6
4. 沙門氏菌(Salmonella choleraesuis, S.C.)6
4.1功能簡介6
4.2 應用6
研究動機8
材料與方法9
1. 材料9
1.1 質體 (Plasmid)9
1.1.1表現載體 (Expression vector)9
1.1.2 RNA干擾質體 (short hairpin RNA, shRNA)9
1.1.3 引子 (primer)10
1.2 抗體 (Antibody, Ab)10
1.2.1 一級抗體 (Primary Ab)10
1.2.2 二級抗體 (Secondary Ab)11
1.3 酶聯免疫吸附試驗試劑11
1.4 實驗動物11
1.5 菌株12
1.6 試劑12
1.6.1細菌培養液12
1.6.2 緩衝液13
1.7 藥物14
2. 實驗方法14
2.1 動物實驗14
2.1.1身分確認14
2.1.1.1 取得小鼠染色體DNA14
2.1.1.2 PCR確認ProT基因型15
2.1.2 IBD動物模式15
2.1.2.1腹瀉評分方式15
2.1.2.2 組織症狀15
2.1.3 野生型和ProT轉殖基因小鼠16
2.1.4 野生型和ProT 基因剔除小鼠16
2.1.5 野生型和Smad7基因剔除小鼠16
2.2 組織固定與包埋17
2.2.1 H&E染色17
2.2.2 免疫組織化學染色法(Immunohistochemistry)17
2.3 酶聯免疫吸附試驗 (ELISA)18
2.4 西方墨點法 (Western blot analysis)19
2.4.1 蛋白樣品製備19
2.4.2 蛋白電泳與免疫轉漬19
2.4.3 免疫沉澱 (Immunoprecipitation, IP)20
2.5 電穿孔法 (Electroporation)20
2.6 統計分析21
結果 22
結論 29
討論 30
參考文獻 32
圖表 38

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