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系統識別號 U0026-1308201216062800
論文名稱(中文) 介白素二十受器抗體在乳癌中的研究
論文名稱(英文) Study of IL-20R1 Antibody on Breast Cancer
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 100
學期 2
出版年 101
研究生(中文) 周毓倫
研究生(英文) Yu-lun Chou
學號 s16994043
學位類別 碩士
語文別 中文
論文頁數 76頁
口試委員 指導教授-張明熙
口試委員-鄭宏祺
口試委員-陳昌熙
中文關鍵字 介白素20  介白素19  乳癌  介白素20受器 
英文關鍵字 IL-19  IL-20  Breast cancer  IL-20R1 
學科別分類
中文摘要 目前乳癌位居女性癌症發生率第一位,死亡率第四位,嚴重威脅婦女健康。介白素-19(IL-19)和介白素-20(IL-20)屬於介白素-10家族的一員,目前已經知這兩分子參與在多種發炎反應相關疾病的病程,然而有研究指出,在腫瘤微環境中的發炎介質,包括細胞激素和趨化因子,可以影響乳癌的進展過程。並且實驗室先前的研究也發現到IL-19和IL-20會在病人腫瘤組織切片中偵測到,本實驗室已分別針對這兩分子在乳癌中扮演的角色,得知IL-19和IL-20與乳癌發展有關聯性。IL-19和IL-20藉由共同受體複合體IL-20 R1/IL-20R2來傳遞訊息 : 為了發展出具有潛力的抗癌藥物,我們製造出針對人類IL-20 R1蛋白的單株抗體,其中之一抗體為51D。實驗上,首先藉由表現不同長度的IL-20 R1蛋白,以用來決定抗原和抗體(51D)的結合位置。而細胞實驗中則搭配使用老鼠乳癌細胞(4T1)和人類乳癌細胞(MDAMB231),在細胞增生和菌落生成能力分析結果發現51D能分別抑制由IL-19所促進的MDAMB231以及IL-20所促進的4T1細胞的生長和菌落生成能力。同時IL-19也會促進MDAMB231細胞的遷徙能力及誘導細胞內發炎物質(TGF-β, IL-1β) 及與轉移相關基因 (CXCR4 MMP2, MMP9)表現量上升,而這些現象皆能藉由加入51D達到抑制的效果。相對而言,IL-20能促進與骨頭破壞有關的因子蛋白酶G、K在4T1細胞裡表達量上升,但其表達量能被51D抑制下來。在動物實驗中也可以觀察到,IL-20 R1的單株抗體,51D,可以阻斷IL-19和IL-20與其受體複合體的結合,進而抑制腫瘤細胞的生長。同時在IL-20 R1的基因剔除鼠與野生鼠的動物實驗中,也能觀察到IL-20 R1基因剔除鼠有較小的腫瘤生成。所以綜合細胞和動物實驗結果,除了再次證明IL-19和IL-20參與乳癌的致病過程,也證明了IL-20 R1的單株抗體具有中和(neutralize) IL-19和IL-20活性的能力,在治療上有極大潛力。
英文摘要 Breast cancer is one of the leading causes of cancer-related mortality in women. Inflammatory mediators, such as cytokines and chemokines in tumor microenvironments affect the progression of breast cancer. Interleukin-19 and Interleukin-20 are cytokines that belong to the IL-10 family. Our previous studies revealed both cytokines are involved in the pathogenesis of breast cancer. Both IL-19 and IL-20 bind to IL-20 R1/IL-20R2 heterodimer to activate signal transduction. To develop a potential drug for treating breast cancer, we have generated the anti-human IL-20 R1 monoclonal antibody to block cytokine function. 51D is one of anti-IL-20 R1 monoclonal antibody. To characterize the antibody, we used the serial deletion of the antigen to determine the binding epitope between 51D and IL-20R1. Boyden chamber assay reveled that IL-19 increased the migration of MDAMB231, a human breast cancer cell line, the activity of which was inhibited by 51D. IL-19 induced the expression of the pro-inflammatory cytokines (TGF-β, IL-1β) and metastasis-associated gene (CXCR4 MMP2, MMP9) in MDAMB231 cells. 51D also inhibited the induction of these genes by IL-19. Furthermore, MTT assays showed that IL-19 and IL-20 promoted the proliferation of MDAMB231 cells and mouse breast cancer cell line 4T1 which was neutralized by 51D. In addition, soft agar colony formation assay showed IL-19 and IL-20 enhanced colony formation of breast cancer cells the activity of which was inhibited by 51D. In vivo, anti- IL-20 R1 antibody, 51D, inhibited MDAMB231 tumor growth. Furthermore, tumor size and tumor weight in the IL-20 R1 knockout mice was smaller than those in the wild type. Our study provides the evidence that 51D can be a potent antagonist to neutralize IL-19 and IL-20 activity.
論文目次 目錄
摘要……………………………………I
Abstract……………………………………II
誌謝………………………………III
目錄……………………………V
圖目錄………………………VIII
附錄目錄……………………IX
縮寫檢索表………………X
第一章 緒論………..1
1-1 腫瘤 (tumor) .....1
1-2 腫瘤微環境 (Microenvironment)…1
1-3 乳癌 (breast cancer) ……………………………………………… 2
1-4 細胞激素 (cytokines) ……………………………………………… 3
1-5 介白素10家族與介白素19和介白素20……………………………………….4
1-6 IL-19和IL-20與其相關疾病………………………6
1-7 乳癌與細胞激素………………………………………… 7
第二章 研究目的……………………………………………………11
第三章 材料與方法……………………………………………………12
3-1 實驗材料…………………………………………………12
3-1-1 細胞株來源及背景…………………………………………12
3-1-2 蛋白質來源…………………………………………………………12
3-1-3 實驗動物來源……………………………………………… 12
3-1-4 實驗隻菌株、質體與培養基...................12
3-1-5 化學物品及特殊實驗用品來源…………………………………………14
3-1-6 各種試劑配置……………………………………………………14
3-1-7 限制酵素……………………………………………………16
3-1-8 實驗使用儀器……………………………………………17
3-2 實驗方法………………………………………………………………18
3-2-1 細胞增生能力分析 (Cell proliferation assay) …………………18
3-2-2西方點墨法 (Western blotting) …………………………………………18
3-2-3細胞遷徙能力分析 (Cell migration assay) ……………………………19
3-2-4 聚落生成Soft Agar colony formation assay....19
3-2-5 同步定量聚合酶連鎖反應…………………………………20
3-2-6 動物實驗………………………………………………………………21
3-2-7 構築人類依序截短型IL-20R1 重組蛋白於pMAL-c2X載體…………21
(A) 聚合酶連鎖反應(Polymer chain reaction ; PCR) ………………22
(B) 製備insert與vector…………………………………………………22
(C) 限制酶處理 (Restriction enzyme digestion) …………………23
(D) 接和反應 (ligation reaction) …………………………………23
(E) 製備E.coli 的勝任細胞 (competent cell) ……………………………24
(F) 形質轉移(transformation) ……………………24
(G) 單一菌落PCR(Colony PCR) ………………………………………24
(H) 利用QIAGEN kit 抽取其質體(plasmid) ………………………24
3-2-8構築人類突變型IL-20R1 重組蛋白於pMAL-c2X載體…………………25
3-2-9 由大腸桿菌系統表現人類截短型及突變型IL-20R1 重組蛋白…25
第四章 結果 ........26
4-1 界定IL-20 receptor 1與其單株抗體51D之抗原決定位 (epitope) ........................26
4-2 51D可以抑制因IL-19及IL-20所促進的細胞增生能力........................................27
4-3 由hIL-19促進的細胞遷徙能力可以被51D給抑制................................................27
4-4 51D可以抑制因IL-19及IL-20所促使的聚落生成(colony formation)能力..........27
4-5 51D抑制IL-19所引起的MMP2和MMP9表達量................................................28
4-6 51D抑制IL-19所引起的CXCR4表達量....................................................28
4-7 51D抑制IL-19所引起的Cathepsin K 和Cathepsin G表達量............................29
4-8 51D抑制IL-19所引起的IL-1β和TGF-β表達量.......................................................29
4-9 IL-20 R1單株抗體能抑制老鼠腫瘤的生長...............................................................30
4-10 IL-20 R1單株抗體能抑制腫瘤中IL-19、IL20和CXCR4基因的表達量............30
4-11 IL-20 R1剔除鼠可以觀察到老鼠腫瘤被抑制的現象.............................................31
4-12 IL-20 R1剔除鼠的腫瘤中IL-19、IL-20、TGF-β、MMP2、MM2 Cathepsin K 和Cathepsin2基因的表達量較野生較少.......... 31
第五章 討論...........................32
參考文獻.............................................38
圖...................................................45
附錄.........................................65
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