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系統識別號 U0026-1408201211541200
論文名稱(中文) 介白素二十受器一之抗體在慢性腎臟疾病中的研究
論文名稱(英文) Study of IL-20 Receptor 1 Antibody in Chronic Kidney Disease
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) 陳薇婷
研究生(英文) Wei-Ting Chen
學號 s16991079
學位類別 碩士
語文別 中文
論文頁數 60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 細胞激素  介白素二十受器  慢性腎臟疾病 
英文關鍵字 Cytokines  IL-20R1  Chronic Kidney Disease 
學科別分類
中文摘要 慢性腎臟疾病是目前全球最為關注的重要健康課題,直至現今仍然無法找出一個有效治療腎臟衰竭的方法。介白素十九(IL-19)與介白素二十(IL-20)屬於介白素十的家族成員,它們共用相同的受器複合物介白素二十受器一與介白素二十受器二(IL-20R1/IL-20R2)來進行下游的訊息傳遞。先前研究指出,IL-19與IL-20參與在許多發炎疾病當中,包含類風溼性關節炎、乾癬以及腎衰竭等。既然IL-19與IL-20同時參與了腎臟疾病的致病過程,因此本篇研究想藉此探討IL-20R1之抗體51D是否針對慢性腎臟疾病具有治療的功效。首先在小鼠集尿管細胞中,可以發現51D能夠抑制IL-19所誘發之MCP-1、TGF-β1、α-SMA、collagen I以及collagen III的mRNA表現以及TGF-β1蛋白質的表達,同樣在人類近曲小管上皮細胞也能看到51D抑制IL-20所誘發的TGF-β1產生。單側尿道阻斷(UUO)是最常拿來研究腎纖維化及腎臟疾病的實驗方法,因此利用UUO的方式建立慢性腎臟疾病的動物模式來驗證IL-20R1之抗體51D的治療效果。我們發現經過UUO處理的小鼠,纖維化相關分子TGF-β1、α-SMA、collagen I以及III表現量明顯增加,而注射IL-20R1之抗體51D後確實有抑制其表現的效果。同樣的將IL-20R1之基因剔除鼠處理過UUO後,亦能發現其能降低TGF-β1、α-SMA以及collagen I的表現,此時便更進一步的證實,IL-20R1之抗體具有治療慢性腎臟疾病的潛力,能夠中和IL-19與IL-20所引起的發炎反應。
英文摘要 Chronic kidney disease (CKD) is a worldwide public health problem. Until now, there have been no effective therapies to halt the renal failure. Interleukin-19 (IL-19) and interleukin-20 (IL-20) are the members of interleukin-10 (IL-10) family. They share the receptor complexes IL-20R1/IL-20R2. Previous studies showed that IL-19 and IL-20 are involved in several inflammatory diseases, such as rheumatoid arthritis, psoriasis and renal failure. Therefore, we aimed to study whether anti-IL-20R1 monoclonal antibody (mAb), 51D have a therapeutic potential for CKD. We found that 51D could inhibit the IL-19-induced monocyte chemotactic protein-1 (MCP1), transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), collagen I, collagen III transcripts and TGF-β1 production in mouse collecting tubular epithelial cell (M1). It also inhibited IL-20-induced TGF-β1 transcripts in human proximal tubular epithelial cell (HK2). Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and considered a model of CKD. To determine the in vivo function of anti-IL-20R1 mAb, we generated a CKD animal model of UUO in mice and treated with anti-IL-20R1 mAb. We found a marked increase of renal fibrosis-related molecule TGF-β1、α-SMA、collagen I and III transcripts and TGF-β1 protein in mice after UUO. Anti-IL-20R1 mAb reduced the TGF-β1、α-SMA、collagen I and III expression. In addition, TGF-β1、α-SMA and collagen I were also significantly decreased in IL-20R1 knock-out mice after UUO. These results suggested that anti-IL-20R1 mAb may have a therapeutic potential for CKD by neutralizing the activities of IL-19 and IL-20.
論文目次 摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 VII
附錄目錄 VIII
縮寫檢索表 IX
第一章 緒論
A. 慢性腎臟疾病(Chronic Kidney Disease, CKD) 1
B. 細胞激素(Cytokines) 2
C. 細胞與慢性腎臟疾病之關係 3
D. 介白素十(Interleukin-10, IL-10)家族 5
E. 介白素二十(Interleukin-20, IL-20)與其相關疾病介紹 5
F. 介白素十九(Interleukin-19, IL-19)與其相關疾病介紹 6
G. 介白素二十受器一(Interleukin-20 receptor 1, IL-20R1)之 單株抗體 7
H. 常見CKD動物模式之建立 8
第二章 研究目的 9
第三章 材料與方法
I.實驗材料
A.細胞、菌種與質體來源 10
B.實驗動物來源 11
C.培養基與緩衝溶液 11
II. 實驗方法
A. 構築人類截短型介白素二十受器一 (IL-20R1)膜外重組蛋白於pMAL/c2X載體 16
a) 聚合酶連鎖反應(Polymer chain reaction, PCR) 16
b) 製備Insert及Vector 17
c) 限制酶處理(Restriction enzyme digestion) 18
d) 接合反應(Ligation reaction) 18
e) 製備勝任細胞(Competent cell) 18
f) 形質轉移(Transformation) 19
g) 單一菌落PCR(colony PCR) 19
h) 利用QIAGEN kit 抽取質體 20
B. 構築人類突變型IL-20R1重組蛋白於pMAL/c2X載體 20
C. 由大腸桿菌系統表現人類截短型(突變型) IL-20R1重組蛋白 21
D. 西方墨點法(Western blotting) 21
E. 酵素連結免疫吸附分析法(Enzyme-linked immunosorbent assay) 21
F. 免疫組織化學染色法(Immunohistochemical staining) 22
G. 細胞實驗 23
H. RNA萃取(extraction) 23
I. 反轉錄酶-聚合酶鏈鎖反應(Reverse transcriptase- polymerase chain reaction, RT-PCR) 23
J. 同步定量聚合酶鏈鎖反應(Real time polymerase chain reaction, Real time PCR) 24
K. 抗體純化 25
a) 收集小鼠腹水 25
b) 純化腹水 25
L. 單側尿道阻斷(Unilateral ureteral obstruction, UUO)25
M. 動物實驗設計 26
第四章 實驗結果
A. 界定IL-20R1與其單株抗體7Gw之抗原決定位(epitope) 27
B. Anti-IL-20R1單株抗體7Gw與51D可抑制人類近曲小管上皮細胞表達TGF-β1 28

C. Anti-IL-20R1單株抗體7Gw與51D可抑制小鼠集尿管細胞表達MCP-1、TGF-β1、α-SMA、collagen I 29
D. Anti-IL-20R1單株抗體7Gw與51D可抑制小鼠集尿管細胞分泌出TGF-β1蛋白 29
E. 單側尿道阻斷(UUO)誘發小鼠慢性腎臟疾病(CKD)動物模式 30
F. Anti-IL-20R1單株抗體51D能夠抑制CKD小鼠表達TGF-β1、α-SMA、collagen I以及collagen III 30
G. Anti-IL-20R1單株抗體51D可以降低CKD小鼠血清中TGF-β1的表現 31
H. IL-20R1基因剔除小鼠建立CKD動物模式 31
I. IL-20R1基因剔除可降低CKD小鼠表達TGF-β1、α-SMA、collagen I 31
J. IL-20R1基因剔除可降低CKD小鼠血清中TGF-β1的表現 32
第五章 討論 33
參考文獻 36
實驗圖表 40
附錄 56
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