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研究生:陳姿伃
研究生(外文):Tzu-Yu Chen
論文名稱:探討鼠傷寒血清型沙門氏菌及現行治療沙門氏菌的抗生Ceftriaxone 對於人類腸道上皮細胞β-防禦素表現之影響
論文名稱(外文):Impacts of Salmonella enterica serovar Typhimurium and antibiotics on human β-defensin expression in human intestinal epithelium
指導教授:方旭偉方旭偉引用關係
口試委員:黃志宏翁文慧方旭彬
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:68
中文關鍵詞:沙門氏菌防禦素人類腸道上皮細胞抗生素
外文關鍵詞:Salmonellalβ-defensinhuman intestinal epithelial cellsantibiotics
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鼠傷寒沙門氏桿菌是引起全世界疾病和死亡的一種重要病原菌。至今,對於
鼠傷寒沙門氏桿菌,其感染人類腸道上皮細胞的早期免疫反應,所知仍然有限。
本研究首先建立LS174T 人類腸道上皮細胞體外培養模式,之後利用鼠傷寒沙門氏
桿菌(Salmonella Typhimurium)野生株SL1344,及缺少SPI-1 基因之突變株△spaS,
感染LS174T 後,以RT-PCR 及ELISA 方法,測量細胞激素IL-8、β-防禦素1、2
和3 之基因與蛋白質表現。而後,更進一步探討臨床上常用,治療鼠傷寒沙門氏
菌桿菌感染之抗生素Ceftriaxone,其對感染後細胞激素IL-8、β-防禦素1、2 和3
之基因與蛋白質表現之影響。
研究結果發現,野生株感染細胞後,可以增加細胞激素IL-8、β-防禦素1、2
和3 之基因表現,而缺少SPI-1 基因突變株,雖然也可以增加表現,但能力較野生
株差。野生株及突變株分別感染細胞後,以抗生素Ceftriaxone 處理細胞,可以抑
制IL-8 之基因表現,但不影響β-防禦素1 基因表現,相反的,卻增加β-防禦素2
和3 之基因表現。雖然Ceftriaxone 影響基因表現,但對於蛋白質表現影響並不顯
著。本研究成果指出,腸道細胞遭遇鼠傷寒沙門氏桿菌感染時,可能增加抗菌基
因表現協助抵抗感染,而治療鼠傷寒沙門氏菌桿菌感染時,投以抗生素Ceftriaxone
並不會干擾此抗菌機制。

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is an
important pathogen which causes morbidity and mortality worldwide. So far, the early
immune responses in human intestinal epithelial cells after Salmonella Typhimurium
infection are little known. In this study, we have established an in vitro model to
observe IL-8、β-defensin 1、2 and 3 gene and protein expression in human intestinal
epithelial cells after Salmonella Typhimurium wild-type strain SL1344 or SPI-1 mutant
strain △ spaS infection by RT-PCR and ELISA assay . Furthermore, the effect of
Ceftriaxone treatment after infection on gene and protein expression of human IL-8、
β-defensin 1、2 and 3 were investigated.
Our results shown the mRNA level of IL-8、β-defensin 1、2 and 3 were increased
after wild-type strain and mutant strain infection. However, the ability of wild-type
strain is higher than mutant strain. The mRNA level of IL-8 after infection was reduced
by Ceftriaxone treatment. However, Ceftriaxone did not affect the mRNA expression
of β-defensin-1. On the contrary, the mRNA level of β-defensin 2 and 3 were
increased. Furthermore, the level of the protein expression affected by Ceftriaxone
treatment was not significant. The results of this study indicated that the intestinal
cells encounting typhimurium Salmonella infection may increase the antimicrobial
gene expression to resist infection, and treatment of Salmonella typhimurium
infection with Ceftriaxone may not interfere with this antibacterial mechanism.

中文摘要 .i
英文摘要 ii
誌謝 iii
目錄 ..iv
表目錄 .vii
圖目錄 viii
第一章 文獻回顧 1
1.1 食源性腸胃疾病 1
1.2 沙門氏菌的簡介 1
1.2.1 歷史回顧 1
1.2.2 型態 2
1.2.3 血清型 2
1.2.4 感染途徑 3
1.2.5 流行病學 3
1.2.6 宿主 4
1.2.7 感染時程 4
1.2.8 感染的臨床症狀 4
1.2.9 感染的免疫反應 5
1.2.10 台灣的非傷寒沙門氏菌感染 5
1.3 人類消化道系統 6
1.3.1 人類消化系統簡介 6
1.3.2 人類腸道對致病菌之防禦機制 6
1.4 防禦素(Defensins) 7
1.4.1 防禦素(Defensins)的簡介 7
1.4.2 β-防禦素 8
1.4.3 人類β-防禦素1 8
1.4.4 人類β-防禦素2 8
1.4.5 人類β-防禦素3 9
1.5 抗生素基本介紹 …9
第二章 研究動機與目的 12
第三章 材料與方法 13
3.1 藥品與藥劑 13
3.2 儀器與設備 14
3.2.1 台北科技大學 14
3.2.2 雙和醫院 15
3.3 實驗步驟 15
3.3.1 配置細胞培養液 15
3.3.2 LS174T 細胞之培養 16
3.3.2.1 解凍LS174T細胞 16
3.3.2.2 繼代 LS174T 細胞之流程 16
3.3.2.3 凍存細胞之流程 16
3.3.2.4 種植 LS174T 細胞之流程 17
3.3.3 LS174T 細胞生長觀察 17
3.3.4 WST-8 assay細胞活性測試 17
3.3.5 Salmonella Typhimurium菌的培養 18
3.3.5.1 SL1344營養成份的配置 18
3.3.5.2 ∆spaS營養成份的配置 18
3.3.5.3 菌的培養 18
3.3.5.4 菌數的評估 19
3.3.6 建立沙門氏菌SL1344、△spaS感染LS174T細胞模式 19
3.3.7 感染SL1344、∆spaS的LS174T細胞之
毒性(Cytotoxicity)分析 21
3.3.7.1 Lactate Dehydrogenase(LDH)分析 21
3.3.7.2 Trypan blue exclusion 存活測試 21
3.3.8 基因表現之評估 22
3.3.8.1 RNA之純化 22
3.3.8.2 反轉錄聚合酶反應
(Reverse Transcription-PCR) 22
3.3.8.3 定量聚合酶鏈鎖反應
(Reverse Transcription-PCR) 23
3.3.9 建立沙門氏菌SL1344、△spaS感染LS174T細胞後
Treated抗生素Ceftriaxone之模式 24
3.3.10 酵素連結免疫吸附分析法 28
3.3.10.1 IL8 酵素連結免疫吸附分析法 28
3.3.10.2 hBD3 酵素連結免疫吸附分析法 29
3.3.11 沙門氏菌SL1344、△spaS感染LS174T細胞之塗盤 30
3.3.11.1感染沙門氏菌並處理Gentamincin抗生素塗盤 30
3.3.11.2感染沙門氏菌並處理Ceftriaxone抗生素塗盤 30

第四章 結果與討論 32
4.1 LS174T 細胞之培養 32
4.1.1 LS174T 細胞型態 32
4.1.2 LS174T細胞生長 33
4.1.3 LS174T細胞生長活性測試 WST-8 assay 34
4.2 菌數的評估 35
4.3 沙門氏菌SL1344、△spaS感染LS174T細胞之
Lactate Dehydrogenase(LDH)分析 36
4.4 沙門氏菌SL1344、△spaS感染LS174T細胞之
Trypan blue exclusion 存活測試 37
4.5 LS174T細胞受到沙門氏菌SL1344、△spaS感染
其defensins基因表現 40
4.6 沙門氏菌SL1344、△spaS感染LS174T細胞後Treated
抗生素Ceftriaxone 之效應 43
4.6.1 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 之效應 43
4.6.2 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 3小時的基因表現 44
4.6.3 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 6小時的細胞毒性分析 47
4.6.4 感染SL1344、△spaS的LS174T細胞並Treated抗生素
Ceftriaxone 6小時的Trypan blue exclusion存活測試 48
4.6.5 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 的IL8蛋白變化量 50
4.6.6 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 的hBD3蛋白變化量 51
4.7 沙門氏菌SL1344、△spaS感染LS174T細胞之塗盤................52
4.7.1 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Gentamincin 1小時後上清液塗盤 52
4.7.2 感染SL1344、△spaS的LS174T細胞並Treated
抗生素Ceftriaxone 3小時、6小時後上清液塗盤 53
第五章 結論 57
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