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系統識別號 U0026-0602201301590200
論文名稱(中文) 實驗性艱難梭狀桿菌腸炎的免疫病理機制之探討
論文名稱(英文) Study on the immunopathogenesis of experimental Clostridium difficile colitis
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 1
出版年 102
研究生(中文) 周柏含
研究生(英文) Po-Han Chou
學號 t36991066
學位類別 碩士
語文別 英文
論文頁數 48頁
口試委員 指導教授-蔡佩珍
口試委員-蔡曜聲
召集委員-詹明修
中文關鍵字 艱難梭狀桿菌  制酸劑  過氧化小體增生活化受體γ  過氧化小體增生活化受體γ促效劑 
英文關鍵字 C. difficile  proton pump inhibitor  PPARγ  PPARγ agonist 
學科別分類
中文摘要 艱難梭狀桿菌感染是目前造成住院病患感染性腹瀉的主要原因,造成的病症以快速及劇烈的腸道發炎反應為主,加上宿主體內不易產生免疫反應用以抵抗細菌入侵,因而造成感染的再發率提升。由於現今對艱難梭狀桿菌感染的病理免疫機制認知之不足,因而成為發展預防及治療性藥物的最大難關。在臨床上已知制酸劑會增加艱難梭狀桿菌的感染機率,故我們藉由制酸劑結合抗生素模式感染野生小鼠,並且利用從人體感染可觀察到的症狀,如:體重減輕、排泄物形態差異、盲腸重量改變以及腸道細菌量改變等,用以判讀小鼠感染後疾病嚴重程度的差異。結果發現結合制酸劑的組別可明顯見到小鼠腹瀉及虛弱,未結合制酸劑組別則沒有明顯的感染症狀產生。而觀察小鼠大腸組織切片染色以及先天免疫相關調控基因,也可發現在感染組別的組織中可明顯看到嗜中性球浸潤以及表皮細胞破損等現象,而發炎激素的基因表現量也隨之上升,這些特徵都隨著制酸劑的給予而增加嚴重度。腸道中的核接受體Peroxisome proliferator-activated receptor γ (PPARγ)及其下游的CD36也因感染而表現量下降,同時也可觀察到受PPARγ調控的抗菌胜肽的表現量改變和杯狀細胞的損壞。PPARγ的轉錄共同活化因子PGC-1α相關的粒線體合成基因也受到影響而降低表現量。另外,可經由PPARγ所調控之Bcl-2表現量也隨之下降,進而我們發現長到細胞凋亡的比率增加。故在此我們假設艱難梭狀桿菌感染是藉由抑制PPARγ進而影響抗菌胜肽及杯狀細胞產制黏液,進一步造成細胞凋亡而造成腸炎之嚴重程度上升。為了證實這一點,我們使用PPARγ的促進劑Pioglitazone來試圖活化PPARγ進而減緩疾病發生,並利用NF-κB冷光報導小鼠監控發炎情形。最後發現藉由促進劑的使用,同時可以降低發炎的程度並且減輕病徵,如減少盲腸重量下降,證明藉由PPARγ作為治療方向將有機會降低艱難梭狀桿菌感染的引發的腸炎症狀。
英文摘要 Clostridium difficile infection (CDI) is currently the leading cause of infectious diarrhea in hospitalized patients. The intense acute inflammatory response appears to be a major factor in causing colonic injury in CDI. An inability to mount a protective immune response to C. difficile appears to underlie susceptibility to recurrent infection. Gaps in our understanding of the immunopathogenesis of CDI present major challenges to the development of better preventive and therapeutic strategies against this problem. Thus, to overcome the resistance of wild-type mice to CDI, we translated our clinical findings to set up antibiotic-proton pump inhibitors (PPI)-associated clostridial colitis in wild-type C57BL/6 mice. We found that no significant signs of CDI were observed in the group without PPI treatment, whereas the group with PPI treatment exhibited diarrhea and signs of weakness. Further, CDI severity was confirmed by measuring clinical signs, such as weight loss, loss of stool consistency, decreased cecum weight, colonic pathology and increased bacterial load, which were shown to mimic CDI on human. Colon histology and the expression of innate immune-related genes were examined and found inflammation was increased, such as neutrophil infiltration, epithelial damage, and inflammatory cytokine genes were increased during CDI, especially after PPI treatment. In addition, the expression of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor prominently expressed in gut, and downstream CD36 was decreased in the colon. Alteration of PPARγ-regulated antimicrobial peptides (AMPs) and goblet cell secreted mucins were observed. PPARγ coactivator-1α (PGC-1α), which was responsible for mitochondrial biogenesis, was also down-regulated. Besides, we also found the expression of PPARγ-regulated Bcl-2 was decreased and apoptotic cells were increased during CDI. Therefore, we hypothesized that down regulation of PPARγ may influence innate immune protective ability, including antimicrobial peptide and mucins, and induce more apoptosis in colon that develop severe CDI. To prove it, we used a PPARγ agonist, Pioglitazone, to protect mice from C. difficile induced inflammation and disease progression through the activation of PPARγ. We monitored the effect of Pioglitazone on CDI by using of NF-κB-dependent luciferase mice. With Pioglitazone treatment, the NF-κB activation-mediated inflammation was decreased and disease progression, loss of ceacum weight, was reversed, implicating PPARγ-targeting therapeutic approaches may pave a way to ameliorate colitis symptoms of CDI.
論文目次 CONTNETS
中文摘要...……………………………………………………………………I
ABSTRACT……………………………………………...………………….II
致謝…………………………………………………………………………III
CONTENTS……………………………………………..………………….IV
INDEX OF TABLES AND FIGURES……………………………….…..VII
INTRODUCTION………………………………………………………...…1
Epidemiology of Clostridium difficile……………………….………..……1
Pathogenesis of Clostridium difficile…………………………………….....2
Animal model………………………………………………………….…....4
Colitis…………………………………………………………………….....5
Colonic PPARγ………………………………………………...….……...…6
PPARγ agonist treatment………………………………………………........7
MATERIALS AND METHODS……………………………………………9
Bacteria strain…………………………………………………………..…..9
Animal…………………………………………………………………....…9
Bacterial culture……………………………………………………….…....9
CDI induced model………………………………………………….…….10
Pioglitazone treatment…………………………………………………......11
In vivo imaging………………………………………………………….…11
CDI symptoms…………………………………………………………….11
Bacterial burden…………………………………………………………...11
Histopathology examination……………...……………….………………12
Immunohistochemistry staining…………………………………………...12
Immunofluorescent staining……………………………………………….12
Tissue RNA extraction…………………………………………………….13
Reverse transcription and real-time PCR………………………………….13
Tissue protein extraction…………………………………………………..14
Western blotting……………………………………………………….......14
In vivo intestinal permeability measurement……………………………...15
Detection of LPS amount………………………………………...………..15
TUNEL assay………………………………………………………….…..15
Data analysis………………………………………………………………15
RESULTS……………………………………………………………….…..16
PPI enhanced the CDI susceptibility………………………………..……..16
C. difficile colonized in mice colon…………………………………..…...17
Histopathological analysis of colonic tissues were performed from CDI mice………………………………………………………………..............17
NF-κB activated mainly in colon after CDI……………………………….17
Pro-inflammatory cytokine expression was up-regulated after CDI……...18
Epithelial integrity was altered after CDI…………………………………19
PPARγ expression decreased in colonic epithelial cells after CDI..............19
Intestinal antimicrobial peptides altered after CDI…………………..........20
Goblet cells depleted after CDI………………………………………........20
Mitochondrial biogenesis was decreased after CDI……………….............21
Bcl-2 reduction led to apoptosis after CDI………………………………..21
PPARγ mediated the disease progression and anti-inflammatory response of Pioglitazone………………………………………………………………..22
DISCUSSION……………………………………………………………….23
REFERENCES……………………………………………………………..28
APPENDIX…………………………………………………………………47
REAGENTS LIST………………………………………………………..48

INDEX OF TABLES AND FIGURES
Table 1. Mouse primer pairs. …………………………………………..….32
Figure 1. Schematics of experimental design. ……………………...…….34
Figure 2. Comparison of CDI disease progression in different treatments. ……………………………………………………………...…..35
Figure 3. Evaluation of C. difficile colonization by culture and amplification of toxin B gene in stool. …………………………………….36
Figure 4. Increased inflammatory signs in colon histology after CDI. …………………………………………………………………….…...37
Figure 5. Colonic NF-κB activation induced by C. difficile. ………….....38
Figure 6. Colonic inflammatory genes expression profile induced by C. difficile. ………………………………………..……………………..….….39
Figure 7. Colonic integrity is disrupted by CDI. ……………………...…40
Figure 8. Expression of colonic PPARγ is decreased in colon after CDI. …………………………………………………………………...….....41
Figure 9. Expression levels of antimicrobial peptides are changed by C. difficile. ……………………………………………………………..…...….42
Figure 10. Colonic goblet cells and mucins are decreased after CDI. .....43
Figure 11. Expression levels of mitochondrial biogenesis genes are decreased after CDI. …………………………..……………………...……44
Figure 12. Colonic apoptosis induced by C. difficile. ……………..….…..45
Figure 13. PPARγ activation induced by Pioglitazone attenuates CDI symptoms and inflammation. ……………..……………………………....46

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