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系統識別號 U0026-0509201205362400
論文名稱(中文) 利用小鼠模式研究含雙色胺酸功能區氧化還原酶在發炎反應中的角色
論文名稱(英文) Study of WW domain-containing oxidoreductase using inflammatory animal models
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 100
學期 2
出版年 101
研究生(中文) 劉勇材
研究生(英文) Yung-Tsai Liou
學號 S46991148
學位類別 碩士
語文別 中文
論文頁數 77頁
口試委員 指導教授-徐麗君
口試委員-林以行
口試委員-張南山
口試委員-詹明修
中文關鍵字 含雙色胺酸功能區氧化還原酶  發炎反應 
英文關鍵字 WW domain-containing oxidoreductase  inflammatory responses 
學科別分類
中文摘要 人類WWOX基因可轉譯出具有抑制腫瘤生成作用的含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase, WWOX),在小鼠又稱為 WOX1 蛋白質。為了研究 WWOX/WOX1 蛋白質在體內的功能,我們實驗室成功地生產出 Wwox-/- 小鼠,而且發現 Wwox-/- 小鼠在出生後一個月內會死亡,顯示 WWOX/WOX1 蛋白質除了具有腫瘤抑制功能外,尚有其他的生理功能。為了研究 WWOX/WOX1 蛋白質在體內的功能,我們利用 Wwox+/+ 與 Wwox+/- 小鼠,建立發炎的實驗動物模式進行研究。我們發現在實驗動物模式中,Wwox+/- 小鼠出現體重急遽下降與死亡的現象。與 Wwox+/+ 小鼠相比之下,Wwox+/- 小鼠脾臟有腫大的情形。此外,在給予處理後的小鼠血清中,也偵測到大量的介白素-6分泌。在實驗動物模式中,小鼠的調控型 T 細胞數量並沒有任何變化,顯示 WWOX/WOX1 蛋白質表現多寡並不影響調控型 T 細胞分化的數量。總結目前的結果,我們推測在小鼠的發炎反應中, WWOX/WOX1 蛋白質扮演重要的調控性角色。
英文摘要 Human WWOX gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase (designed WWOX or murine WOX1). For analyzing the functional roles of WWOX/WOX1 in vivo, we have successfully generated Wwox gene knockout mice in our laboratory. All the whole-body Wwox-/- mice died within a month after birth, suggesting that WWOX/WOX1 functions beyond a tumor suppressor. To investigate the in vivo function of WWOX/WOX1, we tested inflammatory mouse model using Wwox+/+ and Wwox+/- mice. We found that Wwox+/- mice exhibited more severe disease symptoms and higher mortality rate than the wild-type control mice. Compared with Wwox+/+ mice, increased spleen weights were observed in Wwox+/- mice after treatment. High serum levels of interleukin (IL)-6 were detected in treated mice, suggesting that these mice developed more severe inflammatory responses. Comparable numbers of regulatory T cells (Treg) were determined in mice, indicating that WWOX/WOX1 may not affect Treg cell differentiation. Together, our results suggest that WWOX/WOX1 plays important roles in regulating inflammatory responses in mice.
論文目次 中文摘要 I
英文摘要 II
致謝 III
目錄 IV
圖表目錄 VII
中英文縮寫指引 VIII
緒論 1
含雙色胺酸功能區氧化還原酶 (WWOX/WOX1) 1
WWOX/WOX1蛋白質與癌症的關係 1
發炎性腸道疾病 2
研究 IBD 的動物模式 3
多發性硬化症 4
研究 MS 的動物模式 4
IL-17、TH17細胞與發炎相關的疾病 5
研究目的 6
材料與方法 7
A. 材料 7
A-1基因剔除小鼠 7
A-2試劑藥品 7
A-3抗體 8
A-4耗材 9
A-5儀器 10
B. 方法 11
B-1組織細胞培養液 (tissue culture medium) 之製備 11
B-2 MOG35-55-induced experimental autoimmune encephalomyelitis model 12
B-3 DSS-induced colitis model 14
B-4分離小鼠脾臟細胞 15
B-5分離小鼠腸繫膜淋巴結細胞 (mesenteric lymph nodes, MLNs) 16
B-6 MOG35-55 reactivated 脾臟細胞 17
B-7利用 Flow cytometry 分析細胞表面抗原 17
B-8利用 Flow cytometry 偵測 Treg cells (CD4+CD25+Foxp3+) 18
B-9細胞內的細胞激素抗原染色分析 (Intracellular cytokine staining) 19
B-10偵測組織內的DNA sequence 20
B-10-1組織DNA萃取 20
B-10-2 PCR鑑定小鼠基因型 21
B-10-3瓊脂膠體電泳 (DNA agarose gel electrophoresis) 23
B-11組織mRNA表現量的測定 24
B-11-1 Colon RNA萃取 24
B-11-2 RT-PCR實驗 25
B-11-3利用 PCR 進行 cDNA 半定量分析 26
B-11-4定量聚合酶連鎖反應 (real-time PCR or quantitative PCR) 28
B-12利用Kit萃取組織RNA (DSS-induced colitis 的colon sample專用) 30
B-13組織蛋白質表現量測定 31
B-13-1組織蛋白質萃取與定量 31
B-13-2聚丙烯醯胺膠體電泳 (SDS-PAGE) 32
B-13-3 Western blot 34
B-14蘇木紫-伊紅染色法 (Hematoxylin & Eosin stain, H & E stain) 35
B-15統計方法 36
結果 37
Wwox 基因剔除小鼠 (Wwox gene knockout mice) 的產出方式、基因型的鑑定、以及組織中的 WWOX/WOX1 蛋白質表現量 37
在 DSS-induced chronic colitis 實驗模式中,Wwox+/- 小鼠發展出較嚴重的疾病情況 38
小鼠攝入 DSS 後,脾臟以及腸繫膜淋巴結內免疫細胞族群的變化 39
小鼠T細胞中 WWOX/WOX1 蛋白質減少或是不表現時,攝入 DSS 以後也會發展出較嚴重的發炎現象 41
Wwox+/- 小鼠與 Wwoxfl/flCD4Cre 小鼠在 MOG35-55-induced EAE 的動物模式中,會發展出較嚴重的疾病程度 42
Wwox-/- 小鼠 colon 發育不良與自發性的發炎現象 43
討論 45
品系與性別會影響小鼠對於 DSS 的感受性 45
在 DSS-induced colitis 的模式中,WWOX/WOX1 蛋白質對於 macrophages 與其所分泌之細胞激素的調控 46
探討 WWOX/WOX1 蛋白質調控 T 細胞的分化與功能 47
利用 DSS-induced colitis 與 MOG35-55-induced EAE 模式,來探討 WWOX/WOX1 蛋白質功能的延伸研究 49
總結 53
參考文獻 54
圖表 60
附錄 77
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