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研究生:施欣妤
研究生(外文):Hsin-Yu Shih
論文名稱:乳酸菌GG對化療藥物5-氟尿嘧啶誘導人類腸上皮細胞發炎之調節作用
論文名稱(外文):Regulation of 5-Fluorouracil-induced inflammation in human intestinal epithelium by Lactobacillus rhamnosus GG
指導教授:方旭偉方旭偉引用關係方旭彬
指導教授(外文):Hsu-Wei FangShiuh-Bin Fang
口試委員:翁文慧黃志宏
口試日期:2012-07-25
學位類別:碩士
校院名稱:國立臺北科技大學
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:2012
畢業學年度:100
語文別:中文
論文頁數:51
中文關鍵詞:5-氟尿嘧啶乳酸菌GG發炎
外文關鍵詞:5-FluorouracilLactobacillus rhamnosus GGinflammation
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抗癌藥物是常引起系統性免疫抑制及腸道受損,如5-氟尿嘧啶(5-FU)是常見的化療藥物,在臨床上是毒殺腸胃道癌症有較好的治療效果,但常見之副作用為噁心、嘔吐及腹瀉導致體重減輕,嚴重時易引發免疫發炎反應與相關併發症,及抗藥性等問題。化療藥物在對抗癌細胞的同時,也破壞了正常腸道結構,造成腸道黏膜炎,腹瀉等副作用發生,因此在這情況下,發展解決因化學治療製劑產生的副作用是必須的。在過去對於腸胃道疾病的治療,乳酸菌一直被受到注意,因為乳酸菌本身是可促進或減緩腸道的防禦系統功能,如免疫調節作用、免疫減緩作用和免疫排斥反應,且具有抗發炎、調整腸道完整度,以及調控過敏反應等。
因此,在這項實驗中,利用化療藥物5-氟尿嘧啶(5-FU)誘導Caco-2細胞損傷產生發炎反應,並分別以活菌與85℃, 20分鐘的死菌加入於5-FU 誘導Caco-2細胞上的作用,調控發炎反應的能力。初步實驗結果顯示,以2、4小時,MOI=25的活菌與死菌(85℃, 20分鐘)進行毒性分析後,其具有良好生物相容性。最後由基因表現量的結果顯示,經過活菌與死菌經過2小時的作用下,IL-12、TNF-α、MCP-1之mRNA都有明顯向上調控的趨勢;經過4小時的作用,活菌與死菌之間的發炎反應量沒有像在2小時的作用下表現量這麼高,其原因可能是因為在4時間的作用下細胞似乎隨著時間的延長受到不同型式乳酸菌的影響促進的發炎反應趨勢會緩緩向下調控。

Many anti-cancer medicine usually cause system immune inhibition and impaired gut, such as 5-Fluorouracil (5-FU) is a common chemotherapy drug. 5-FU cytotoxicity in clinical gastrointestinal cancers is better therapeutic. It still has common side effects which are nausea, vomiting and diarrhea lead to body weight lose. Chemotherapy drugs also damage the normal intestinal structure, causing intestinal mucous, diarrhea and other side effects occurred in the fight against cancer at the same time. It is impassion serious inflammation and immune-related complications and drug resistance. It’s necessary to reduce side effect of immune inhibition. Therefore, in this case, it’s necessary that development to solve side effects of chemotherapy drugs. In the past, for the treatment of gastrointestinal diseases that lactobacilli has been received attention, because lactobacilli can promote or slow down the intestinal defense system functions, such as immune regulation immune to slow down the role of immune rejection, and has anti-inflammatory, regulate intestinal integrity, and regulation of allergic reactions.
In this study, Caco-2 cells were induced inflammatory response by 5-FU and were treated with the live LGG or heat killed LGG (at 85℃ for 20 min) thereafter the effect to regulate inflammatory responses. These data showed the live LGG and heat killed LGG at an MOI of 25 for 2 and 4 hours treating times that exerted good biocompatibility via the cytotoxicity test. The results of gene expression indicated that after 2 hours live LGG and heat killed LGG treatment, the mRNA expression of IL-12, TNF-α, MCP-1 were significantly up-regulation; after 4 hours treatment, expression of these inflammatory mRNA were lower than 2 hours treatment. We considered, the host cells may be influenced by different types of LGG under 4 hours treatment and inflammatory response was slightly down-regulated.


中文摘要 .i
英文摘要 ii
目錄 .iv
表目錄 .vi
圖目錄 vii
第一章 緒論 1
1.1 前言 1
1.2 實驗動機與目的 2
1.3 實驗設計流程 4
第二章 文獻回顧 5
2.1 腸道結構與功能 5
2.1.1 腸道上皮細胞之生理功能 5
2.1.2 腺窩-絨毛軸 6
2.1.3 大腸直腸癌之發生 6
2.1.4 化療藥物對腸道的影響 7
2.1.5 化療藥物 5-Fluorouracil 8
2.2 人體腸道菌 9
2.2.1 腸道中的細菌數 9
2.2.2 腸道菌相 10
2.2.3 影響腸道菌相因子 10
2.3 益生菌 10
2.3.1 益生菌定義 10
2.3.2 益生菌特性 11
2.3.3 益生菌在腸道中的作用機制 13
2.3.4 益生菌的生理功能 14
2.3.5 益生菌的吸附機制 15
第三章 材料與方法 16
3.1 藥品與試劑 16
3.2 儀器設備 18
3.3 Caco-2細胞培養方法 19
3.3.1 建立Caco-2細胞 19
3.3.2 冷凍細胞活化 20
3.3.3 細胞繼代培養 20
3.3.4 細胞冷凍保存 21
3.3.5 細胞計數 21
3.4 建立化療藥物5-FU誘導Caco-2細胞損傷模式 21
3.4.1 MTT之細胞活性測試 22
3.4.2 化療藥物5-FU誘導Caco-2細胞發炎 (Inflammatory) 22
3.4.3 化療藥物5-FU 誘導腸修復之細胞毒性(Cytotoxicity)
分析 23
3.5 細菌培養方法 24
3.5.1 菌株 24
3.5.2 菌種培養與保存 24
3.5.3 菌株培養基 24
3.5.4 製備不同型式Lactobacillus rhamnosus GG 24
3.5.5 不同型式Lactobacillus rhamnosus GG型態評估 25
3.6 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸修復之
細胞毒性分析 25
3.6.1 Lactate Dehydrogenase(LDH)分析 26
3.7 基因表現之評估 27
3.7.1 RNA之純化 27
3.7.2 反轉錄聚合酶反應(Reverse Transcription-PCR) 27
3.7.3 定量聚合酶鏈鎖反應(Quantifcation Real-Time PCR) 28
3.8 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸發炎
基因表現效應 30
第四章 結果與討論 31
4.1 Caco-2細胞培養方法 31
4.1.1 建立Caco-2細胞 31
4.2 建立化療藥物5-FU誘導Caco-2細胞損傷模型 33
4.2.1 MTT之細胞活性測試 33
4.2.2 化療藥物5-FU誘導Caco-2細胞發炎 (Inflammatory) 34
4.2.3 化療藥物5-FU 誘導腸修復之細胞毒性(Cytotoxicity)
分析 35
4.3 細菌培養方法 36
4.3.1 製備不同型式Lactobacillus rhamnosus GG 36
4.3.2 不同型式之Lactobacillus rhamnosus GG型態評估 38
4.4 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸修復之
細胞毒性(Cytotoxicity)分析 40
4.4.1 Lactate Dehydrogenase(LDH)分析 40
4.5 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸發炎
基因表現之評估 42
第五章 結論 46
參考文獻 47


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