臺灣博碩士論文加值系統

化療所引起的腸道黏膜炎其主要症狀為體重下降、腹瀉、腸道損傷發炎。介白素-19和介白素-20是介白素-10家族成員中的一員，由文獻已知介白素-19和介白素-20皆參與在許多發炎性相關的疾病當中。我們以5-FU這個常見的化療藥物去誘導腸道黏膜炎小鼠動物模式，首先我們利用anti-IL-20單株抗體7E作為治療化療藥物5-FU引起的腸道黏膜炎的小鼠，但7E卻沒有任何的保護效果，因此我們進一步利用IL-20R1剔除小鼠將IL-20的接受器剔除，發現在誘導成腸道黏膜炎時，IL-20R1剔除小鼠可以保護大腸不受到化療藥物的損傷、發炎基因表現量也降低，細胞凋亡的情形在IL-20R1剔除小鼠中也有明顯減緩的情況。接下來我們利用anti-IL-20R1單株抗體51D作為藥物治療5-FU引起的腸道黏膜炎的小鼠，與IL-20R1剔除小鼠的結果一致，51D也可以保護大腸的組織型態、減少發炎基因的表現以及減緩細胞凋亡的情形，結果顯示阻斷IL-20R1可以改善化療所引起的腸道黏膜炎，因此我們認為可能不是IL-20參與在其中而是同樣會接上IL-20R1的IL-19參與在化療所引起的腸道黏膜炎當中。接下來我們利用抗IL-19抗體1BB1作為藥物治療5-FU引起的腸道黏膜炎的大鼠，與51D的結果相似，1BB1可以降低大腸發炎基因的表現，並且可以減少化療藥物對於大腸組織的損傷。因此我們實驗結果顯示IL-19的確作為一個促發炎反應的細胞激素參與在化療引起的腸道黏膜炎當中，並且1BB1阻斷IL-19可能是改善化療引起的腸道黏膜炎的治療藥物。

Chemotherapy-induced intestinal mucositis is a common side effect and a major cause of morbidity and mortality in cancer patients. 5-Fluorouracil (5-FU) is an anticancer drug that is widely used in the treatment of gastrointestinal malignancies. About 50- 80% of patients who received 5-FU resulted in severe mucositis indicated by diarrhea, bloody stool, shortened colon, and infiltration of inflammatory cytokines, such as interleukin-1(IL-1), IL-6 and TNF. IL-20 is a proinflammatory cytokine, which can induce IL-1, IL-6 and TNF in different types of cells. Therefore, we were aimed to investigate the association of IL-20 with chemotherapy-induced intestinal mucositis. We chose the well-established 5-FU-induced intestinal mucositis as the animal disease model. The expression of IL-20 was elevated in colonic tissue of 5-FU-treated mice compared to healthy control analyzed by RT-qPCR and Immunohistochemistry (IHC). However, anti-IL-20 monoclonal antibody 7E did not suppress the symptom of mucositis. In addition, apoptosis in colon was attenuated in IL-20R1KO mice and anti-IL-20R1 antibody(51D)-treated mice. IL-20 R1KO mice and 51D-treated mice also protected the contents of mucin and colon morphology from 5-FU-treated injuries. Thus, we hypothesized that IL-19, which is a proinflammatory cytokine binding to IL-20R1, may be involved in 5-FU-induced intestinal mucositis. We further used anti-IL-19 antibody (1BB1) in rat model to confirm our hypothesis. 1BB1 treatment reduced colon inflammation by inhibiting the inflammatory cytokines. Furthermore, submucosal edema in colon was attenuated in 1BB1-treated rat. Our data indicated that IL-19 was involved in the pathogenesis of 5-FU-induced intestinal mucositis. Therefore, IL-19 antagonist may be a potential therapeutic.

摘要...I
Abstract...II
誌謝...VI
目錄...VII
圖目錄...IX
附錄目錄...X
縮寫檢索表...XI
第一章 緒論...1
1-1 化療所引起的腸道黏膜炎...1
1-2 細胞激素(Cytokines)...1
1-3 細胞激素接受器(Cytokines Receptor)...2
1-4 介白素-10 (Interleukin-10)家族...2
1-5 介白素-19 (Interleukin-19)...3
1-6 介白素-20 (Interleukin-20)...4
1-7 細胞激素與化療引起的腸道黏膜炎...4
第二章 研究動機與目的...5
第三章 實驗材料與方法...7
3-1 實驗材料...7
3-1-1 實驗動物來源...7
3-1-2 細胞來源...7
3-1-3 蛋白質及抗體來源...7
3-1-4 化療藥物來源...8
3-1-5 實驗之試劑配置...8
3-1-6 實驗溶劑及染色劑...11
3-2 實驗方法...11
3-2-1 5-Flurourail 製備...11
3-2-2 動物實驗...11
3-2-3 腹瀉程度觀察...12
3-2-4 免疫組織化學染色法(Immunohistochemistry staining ; IHC)...13
3-2-5 同步定量聚合酶連鎖反應...13
3-2-6 Periodic Acid Schiff staining (PAS staining)...14
3-2-7 DeadEnd™ Fluorometric TUNEL System...14
3-2-8 細胞內訊息傳遞分析(Cellular signal transduction)...15
3-2-9 西方點墨法(Western blotting)...15
第四章 實驗結果...16
4-1 5-FU 誘導腸道黏膜炎小鼠動物模式有IL-20 的高表現量...16
4-1-1 腸道黏膜炎小鼠動物模式建立...16
4-1-2 5-FU 引起的腸道黏膜炎會破壞大腸組織的型態...16
4-1-3 5-FU 引起的腸道黏膜炎會促使IL-20＆IL-20R1 表現量增加...16
4-2 抗IL-20 單株抗體7E 無法減緩5-FU 引起的腸道黏膜炎...17
4-2-1 抗IL-20 單株抗體7E 無法減緩5-FU 引起的腸道黏膜炎的症狀...17
4-2-2 抗IL-20 單株抗體7E 無法減緩5-FU 引起的腸道黏膜炎的大腸組織損傷...17
4-2-3 抗IL-20 單株抗體7E 無法抑制5-FU 引起的腸道黏膜炎發炎細胞激素的基因表現量...18
4-3 IL-20R1 knockout mice 對於5-FU 引起的腸道黏膜炎的保護效果...18
4-3-1 IL-20R1 knockout mice 減緩5-FU 引起的腸道黏膜炎的症狀...18
4-3-2 IL-20R1 knockout mice 改善了腸道黏膜炎所破壞的大腸組織型態...19
4-3-3 IL-20R1 knockout mice 降低IL-1β、TNF-α 的基因表現量...19
4-3-4 IL-20R1 knockout mice 降低ICAM-1、VCAM-1 基因表現...19
4-3-5 IL-20R1 knockout mice 改善了腸道黏膜炎所引起的細胞凋亡...20
4-4 抗IL-20 R1 單株抗體51D 對於5-FU 引起的腸道黏膜炎的保護效果...20
4-4-1 抗IL-20 R1 單株抗體51D 減緩5-FU 引起的腸道黏膜炎症狀...20
4-4-2 抗IL-20 R1 單株抗體51D 對於腸道黏膜炎的大腸組織型態有保護的效果...21
4-4-3 抗IL-20 R1 單株抗體51D 改善了腸道黏膜炎所引起的細胞凋亡...21
4-4-4 抗IL-20 R1 單株抗體51D 降低IL-1β、IL-6 的基因表現量...22
4-4-5 抗IL-20 R1 單株抗體51D 降低ICAM-1、VCAM-1 基因表現...22
4-5 抗IL-19 抗體1BB1 對於5-FU 引起的腸道黏膜炎的保護效果...22
4-5-1 5-FU 引起的腸道黏膜炎會促使IL-19 表現量增加...23
4-5-2 抗IL-19 抗體1BB1 無法減緩5-FU 引起的腸道黏膜炎症狀...23
4-5-3 抗IL-19 抗體1BB1 對於大腸組織型態有保護的效果...24
4-5-4 抗IL-19 抗體1BB1 減緩了腸道黏膜炎所引起的細胞凋亡...24
4-5-5 抗IL-19 抗體1BB1 降低IL-1β、TNF-α、iNOS、TGF-β 的基因表現量...25
4-6 觀察IL-19 在大腸癌細胞的作用...25
4-6-1 Caco-2, HCT-116 細胞中有表現IL-19 及其receptor...25
4-6-2 IL-19 促進Caco-2, HCT-116 表現IL-1β 和 TNF-α...25
4-6-3 IL-19 促進HCT-116 的NF-κB 與Akt 的訊息傳遞蛋白的磷酸化...26
第五章 討論...27
參考文獻...31
圖...34
附錄...55

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