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研究生:蔡佳容
研究生(外文):Jia-Rung Tsai
論文名稱:建立腸道模型平台檢測藥物之免疫調節活性
論文名稱(外文):Establishment the Intestinal Model Platform to Evaluate the Absorption and Immune Regulation of Drugs
指導教授:葉怡玲葉怡玲引用關係
指導教授(外文):Yi-Ling Ye
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:77
中文關鍵詞:IBD免疫抑制劑腸道吸收系統腸黏膜巨噬細胞
外文關鍵詞:IBDimmunomodulatorsintestinal absorption modelintestinal mucosamacrophages
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近年來越來越多研究利用體外Caco-2 腸道上皮細胞transwell模型去模擬口服藥物吸收的情況。而最新研究指出在混合適當比例的HT-29 (杯狀細胞)與Caco-2的細胞模型對於胰島素吸收將比Caco-2細胞系統更接近真實腸道藥物吸收情況。
發炎性腸道疾病 (Inflammatory bowel disease ,IBD )的病患腸道上皮細胞與細胞間的通透性會增加而導致上皮損傷和潰爛且無法防禦細菌的入侵。IBD病患的免疫細胞會滲透至固有層,其中,單核球可被趨化激素由周邊血趨化至發炎部位,轉變成巨噬細胞,且大量分泌促發炎細胞激素,這些激素可促進黏膜發炎程度。為了要探討腸道內藥物與巨噬細胞間的交互作用,我們建立一個腸道細胞與巨噬細胞共培養的腸道模型平台,檢測藥物是否可抑制巨噬細胞分泌的發炎細胞激素,進而達到改善IBD病患的發炎情形。
在實驗設計上,使用腸道模型與免疫細胞的共培養的方式有兩種,第一種是將Caco-2與HC19 (Caco-2/HT-29) 加入藥物(包含桂花粗萃物(Osmanthus Fragrans extract , OFE)、verbascoside (VB) 以及prednisolone培養後的上清液收集以不同稀釋倍數稀釋後再加入至巨噬細胞培養中,觀察巨噬細胞的細胞激素分泌變化;第二種則利用transwell系統讓分化後腸道上皮單層細胞 (monolayer) 與巨噬細胞共培養,觀察藥物刺激腸道細胞後分泌的細胞激素及藥物經腸道細胞吸收後如何影響巨噬細胞。
結果顯示在單細胞培養實驗中,活化態巨噬細胞與OFE 、VB或prednisolone培養下, 只有prednisolone能有效抑制巨噬細胞分泌的促發炎細胞激素IL-8與TNF-α,而OFE 和VB則無此抑制能力;上清液培養法結果顯示,Caco-2上清液會抑制巨噬細胞所分泌的IL-8,但不影響TNF-α表現,HC19上清液會抑制巨噬細胞所分泌的IL-8,促進TNF-α分泌;而在transwell共培養時在,Caco-2細胞模型OFE 、VB以及prednisolone均能抑制活化巨噬細胞所分泌的促發炎細胞激素IL-8與TNF-α,在HC19細胞模型中,prednisolone能抑制活化巨噬細胞所分泌的促發炎細胞激素IL-8與TNF-α,OFE 和VB可抑制IL-8,但OFE 和VB對於TNF-α則沒有效果。
由實驗結果可以看到單細胞培養與共培養間的差異,且在文獻中指出,利用VB治療IBD動物實驗中,VB並無抑制腸道中TNF-α的現象,這與HC19細胞模型看到的結果一致。總結來說,腸道細胞與巨噬細胞共培養的腸道模型平台有三種優勢:第一、能分析腸道內藥物吸收的生物利用度和代謝產物,第二、 能供一個腸道環境評估藥物吸收後免疫細胞的免疫調節活性,第三、探討腸道細胞和免疫細胞間的分子作用機制。未來可藉由此共培養系統篩選抗發炎藥物,用於治療或預防IBD。


During the last few years, using Caco-2 cell (intestinal epithelial cell lines) in vitro system is one popular method to mimic the small intestinal absorption of drugs. Resent research indicates that the insulin absorption pattern of HT-29 (goblet cells) and Caco-2 mixed culture is more closer to the true intestinal absorption situation.
Inflammatory bowel disease (IBD) may present with the following disorder in bowel including: increased the epithelial cell–cell (paracellular space) permeability, bacterial invasion sensitivity, epithelial damage, and ulceration. At the same time, inflamed lamina propria chemoattracted immune cells and locally secreted high levels of proinflammatory cytokines, including IL-8, TNF-αetc. For example, monocytes can be chemoattracted from the peripheral blood to the site of inflammation. At this site, monocyte differentiated into macrophages. Release proinflammatory cytokines by macrophage all contribute to the severity of mucosal inflammation. In order to study the interaction between the drug and macrophages in intestinal environment, we established an intestinal cells and macrophages co-cultured system.
In our experimental design, two cell culture system was evaluated, including: 1. The supernatants from Caco-2 or HC19 (Caco-2/HT-29) culture with or without drugs (Osmanthus Fragrans extract (OFE) , verbascoside, and prednisolon ) were added to the immune cells. The cytokine profile of macrophage was observed; 2. Using transwells to mimic the intestinal mucosal environment, the cytokine profile of macrophage was observed. 3. Using UPLC-MS/MS drug analysis system, the drug absorption and metabolism result could be evaluated.
Prednisolone but not OFE and VB treated activated macrophage secrete lower levels of proinflammatory cytokine IL-8 and TNF-α, In the transwell co-culture system, the secretion of IL-8 and TNF-α was inhibited in the Caco-2 system of activated macrophage. However, the response was different between the two co-culture system. Prednisolone led to a decrease of the macrophage mediators IL-8 and TNF-α, in the HC19 cell type co-culture setup.OFE and VB for HC19 cell type co-culture setup can inhibition of IL-8 but not TNF-α.
There are three advantages of this model including: 1.We can evaluate the drug bioavailable ability and analyze metabolite upon the intestinal monolayer system. 2. Provide one intestinal environment to evaluate the immunomodulatory activity of immune cells after drug absorption. 3. To study the complicated interaction between intestinal cells and immune cells. In the future, transwell co-culture system could be a good model for selecting for the anti-inflammatory drugs that can be used to treat or prevent IBD.

中文摘要..................................................i
英文摘要..................................................iii
誌謝.....................................................vi
目錄.....................................................vii
表目錄...................................................xi
圖目錄...................................................xii
縮寫對照表................................................xiv
第一章 前言及文獻回顧.............................1
1.1藥物口服吸收.........................................1
1.1.1藥物口服吸收系統..............................1
1.1.2 腸道系統介紹....................................2
1.1.2.1 腸道系統組成..................................2
1.1.2.2 腸道內免疫調節...............................3
1.1.2.2.1 發炎調節情形................................4
1.1.2.2.2免疫耐受情形................................5
1.1.3 In inflammatory bowel (IBD)................6
1.1.3.1 治療IBD藥物介紹............................6
1.1.3.1.1 西藥......................6
1.1.3.1.2中藥.................................8
1.1.4 口服藥物吸收研究進展...................................9
2.1 國內外專利檢索.................................10
第二章 研究目的....................................12
第三章 材料與方法...................................14
3.1材料....................................14
3.1.1藥品...........................................14
3.1.2儀器........................................15
3.1.3 細胞株.......................................15
3.1.4細胞培養基組成配法..................16
3.2實驗方法...................................16
3.2.1 細胞培養.....................................16
3.2.1.1繼代培養...........................16
3.2.1.2腸道模型細胞單層培養..........................17
3.2.2 細胞存活率分析..................................18
3.2.3 酵素免疫分析法...............................19
3.2.4腸道模型細胞單層完整性測試..................20
3.2.4.1 TEER..........................................20
3.2.4.2 酚紅滲透性實驗.........................20
3.3 數據分析..............................................22
第四章 實驗結果.......................................23
4.1 細胞存活率分析......................................23
4. 2腸道模型細胞單層完整性測試.............23
4. 2.1 細胞單層完整性分析 (TEER)........23
4. 2.2 酚紅滲透性實驗................................24
4.3 細胞激素表現........................................24
4.3.1 單細胞培養..........................................24
4.3.1.1腸道細胞IL-8分析............................24
4.3.1.2巨噬細胞IL-8及TNF-α分析............24
4.3.2以上清液培養細胞之試驗...................25
4.3.3 Transwell 共培養系統...........................25
4.3.3.1藥物對腸道細胞-巨噬細胞共培養IL-8及TNF-α分泌之影響...... ………….25
4.3.3.2藥物對腸道細胞-激活巨噬細胞共培養IL-8及TNF-α分泌之影響..........26
4.3.3.3 LPS對IL-8及TNF-α分泌之影響.........................27
第五章 討論與結論......................................28
參考文獻...........................................32
實驗結果附表...............................................38
實驗結果附圖.............................................44
英文論文大綱................................................67
作者簡歷...........................................77


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