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研究生:柯星羽
研究生(外文):Hsin-Yu Ko
論文名稱:土肉桂萃取物對於胃幽門螺旋桿菌與受感染人類胃上皮細胞之抗微生物與抗發炎效應
論文名稱(外文):The Anti-Microbial and Anti-Inflammatory Effects of Indigenous Cinnamon Extract on Helicobacter pylori and Helicobacter pylori-infected Human Gastric Epithelium.
指導教授:方旭偉方旭偉引用關係方旭彬
口試委員:翁文惠黃志宏
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:英文
論文頁數:87
中文關鍵詞:土肉桂胃幽門螺旋桿菌人類胃上皮細胞反式肉桂醛IL-8
外文關鍵詞:Indigenous cinnamonH. pyloritrans-cinnamaldehydeAGSIL-8
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胃幽門螺旋桿菌是人類最常見的病原菌之一,CagA (細胞毒素-相關基因 A)為其致病因子,它會在被感染的細胞中誘導促發炎因子IL-8的分泌並刺激持續性的發炎反應。而過度的IL-8的表現被認為是一個會導致慢性消化道疾病像是胃發炎、消化性潰瘍和胃癌等的危險因子。土肉桂已被研究出具有抑制多種細菌以及減緩宿主細胞發炎的能力。我們以水蒸餾法得到土肉桂萃取物後,再利用高效液相層析儀(HPLC)測定其反式肉桂醛濃度為0.12%。在這項研究中,利用胃幽門螺旋桿菌感染人類胃上皮細胞 (AGS cells),接著再加入土肉桂萃取物去作用。評估土肉桂萃取物對於抑制胃幽門螺旋桿菌生長以及抑制其誘導之發炎反應的所利用的方法如下:最低抑制濃度測試(MIC test)、cagA基因表現量測定、IL-8基因表現量測定與促發炎因子IL-8 蛋白質表現量分析(human IL-8 ELISA assay)。基因與蛋白質表現量皆會在短時間與長時間的發炎誘導試驗中被測定。這些實驗結果顯示,土肉桂萃取物之最低抑制胃幽門螺旋桿菌的反式肉桂醛濃度為0.001563%,而在加入反式肉桂醛濃度0.001563%-0.12%的土肉桂萃取物(t-ICE)之組別其抑制胃幽門螺旋桿菌的效果在80%以上。在短時間試驗中,加入土肉桂萃取物治療的組別與不加入土肉桂萃取物的組別相比,其cagA和IL-8的基因表現都有被抑制的趨勢,而促發炎因子IL-8的蛋白質表現也有被降低下來。在長時間試驗中,由幽門桿菌感染及IL-1β刺激所誘發的IL-8的基因與蛋白質表現在有加入土肉桂萃取物治療的組別中也有被抑制下來的現象。這些結果證實,土肉桂萃取物能夠有效地抑制胃幽門螺旋桿菌的生長並且抑制其造成的宿主細胞發炎反應。因此,未來有希望利用土肉桂萃取物來有效地減緩胃幽門螺旋桿菌所造成的消化道疾病,減少、輔助或取代抗生素的使用,以解決現階段胃幽門螺旋桿菌日益嚴重的抗藥性的問題。

Helicobacter pylori (H. pylori) is one of the most common pathogens in humans. CagA (cytotoxic-associated gene A), a virulence factor of H. pylori, induces pro-inflammatory cytokine IL-8 secretion and activates persistent inflammatory responses in infected gastric epithelial cells. Over expression of IL-8 is believed to be an important risk to lead chronic peptic diseases such as gastritis, peptic ulcer, and gastric cancer. Indigenous cinnamon (IC) was reported to be able to inhibit many kinds of bacteria and decrease host cell inflammation. The water distillation method was adopted to extract indigenous cinnamon and the concentration of trans-cinnamaldehyde detected by high-performance liquid chromatography (HPLC) is 0.12%. In this study, human gastric adenocarcinoma cells (AGS cells) were infected with H. pylori then were treated with indigenous cinnamon extract (ICE). To evaluate the inhibitory effects of ICE on H. pylori growth and the suppressive effect of ICE on H. pylori-induced inflammation, the MIC test, cagA gene expression quantities, IL-8 gene expression and human IL-8 ELISA quantities were performed. The gene and protein expression were investigated in the short time and long times inflammation induction trials. These data showed that, the minimum inhibitory concentration of trans-cinnamaldehyde of ICE (t-ICE) was 0.001563% and 0.001563%-0.12% t-ICE achieved as high as 80% of H. pylori inhibition rates in the MIC test. In the short time trials, the ICE treated groups had the effect to inhibit IL-8 and cagA gene expressions. Similarly, IL-8 protein expression was suppressed, too. In the long time trials, H. pylori and IL-1β stimulated IL-8 gene and protein expressions were inhibited with the ICE treating groups. In summary, these results demonstrated that indigenous cinnamon extract exerts a significant effect to inhibit H. pylori growth and H. pylori/ IL-1β-induced inflammatory responses in host cells. Hence, indigenous cinnamon extract could potentially alleviate H. pylori-induced peptic diseases. It may hopefully decrease, facilitate or replace the use of antibiotics to solve the worsening problem in antibiotic resistance of H. pylori.

摘 要 I
ABSTRACT III
ACKNOWLEDGEMENTS V
CONTENTS IX
LIST OF TABLES XII
LIST OF FIGURES XIII
CHAPTER 1 INTRODUCTION 1
1.1. INTRODUCTION OF HELICOBACTE R PYLORI 1
1.1.1. History and Microbiology 1
1.1.2. Epidemiology 2
1.1.3. Virulence Factors of Helicobacter pylori 3
1.1.3.1. cag PAI and Type IV Secretion System 3
1.1.3.2. CagA Protein 4
1.1.3.3. VacA Protein 5
1.1.3.4. Urease 6
1.1.4. Helicobacter pylori Infections Cause Chronic Peptic Disease 9
1.2. Cinnamon and Indigenous Cinnamon 10
1.3. AGS Cells 13
1.4. IL-1β and IL-8 15
1.5. Objective 17
CHAPTER 2 MATERIALS AND METHODS 20
2.1. List of Equipments, Materials and Reagents 20
2.2. Helicobacter pylori Strains and Culture Conditions 21
2.3. AGS Cells and Culture Conditions 22
2.4. Water Distillation to Obtain Indigenous Cinnamon Extract 23
2.5. HPLC Analysis 24
2.6. Short-duration Infection Trial 28
2.7. Long-duration Infection Trial 29
2.8. Cytotoxicity Test 31
2.8.1. LDH Test 31
2.8.2. CCK-8 Test 33
2.9. Measurement of IL-8 Gene Expression Levels by qPCR Assay 35
2.10. Measurement of IL-8 Expression Levels by ELISA Assay 41
2.11. MIC (Minimum Inhibitory Concentration) Test 45
2.12. Statistical Analysis 46
CHAPTER 3 RESULTS AND DISCUSSION 47
3.1. HPLC Analysis 47
3.2. MIC (Minimum Inhibitory Concentration) Test 49
3.3. H. pylori Inhibition Effect 50
3.4. Short-duration Trial 51
3.4.1. Cytotoxicity Test 51
3.4.1.1. Cell Activity Test by CCK-8 51
3.4.1.2. Cytotoxicity Test by LDH 54
3.4.2. qPCR Assay for Gene Expression 56
3.4.3. ELISA Assay for Human IL-8 Protein Expression 59
3.5. Long-duration Trial 60
3.5.1. Cytotoxicity Test 60
3.5.1.1. LDH Test 60
3.5.2. qPCR Assay for Gene Expression 63
3.5.3. ELISA Assay for Human IL-8 Protein Expression 68
CHAPTER 4 CONCLUSIONS 75
REFERENCES 79



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