2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648548摘要：炎性腸症包括克隆氏症及潰瘍性腸炎。在歐美西方先進國家，這是胃腸科最重要的慢性疾病。但在台灣仍然鮮為人知，即使醫師也不熟悉。這幾年的研究發現克隆氏症的發生率在台灣逐年上升，顯示台灣可能因為環境西化或其他因素，造成克隆氏症的重要性逐漸受到重視，本研究也將集中專注於克隆氏症。克隆氏症的致病機轉相當複雜，至今仍不清楚，極可能牽涉基因、腸道內細菌互相作用， 再造成腸道細胞的發炎或凋亡。NOD2/CARD15是克隆氏症最早被發現相關的基因，但是這個基因主要是和歐美的克隆氏症族群有關，可是我們在亞洲族群，包括日本、韓國、香港以及台灣都並未發現這個基因的相關性。近年來韓國的研究顯示IL23R與 TNFSF15這兩個基因與克隆氏症相關，我們初步小規模的研究 也顯示ATG16L1 與TNFSF15這兩個基因可能值得探究；同時我們還可以探討其他細胞激素基因多型性，並將其與臨床症狀互相對照。腸內菌一般認為與克隆氏症致病機轉有關，但是究竟是何種細菌造成克隆氏症的發生仍然不明。抑或是細菌透過脂多醣體(lipopolysaccharide) 的產生，並結合腸道表皮細胞的TLR4受體與 CD14一起來驅動腸道的發炎反應。克隆氏症的病人有可能其TLR4受體突變，造成過度發炎反應， 亦即克隆氏症的產生。本研究的目的即在闡明基因、腸道內細菌交互作用於腸道表皮細胞以致一連串發炎或細胞凋亡的步驟。我們假設一定劑量的腸道內細菌會引起某些特定基因型的人產生腸道表皮細胞的過度發炎。 我們將根據臨床表徵、影像檢查、內視鏡檢查、以及病理切片的結果來做克隆氏症的診斷。同時收集兒科及成人病人。所有病人的白血球將收集化驗以做NOD2/CARD15, ATG16L1, TNFSF2, TNFSF15基因多型性分析。我們也將在病人不同的疾病階段收集他們的糞便，這些糞便檢體將使用聚合酶鏈鎖反應配合變性梯度膠電泳的方法予以分析。主要是利用針對細菌16s rDNA設計共通引子，先做聚合酶鏈鎖反應放大細菌的DNA，再利用變性梯度膠電泳分離各個不同的細菌的DNA ，最後再將電泳分離的DNA序列判讀以辨認究竟是何細菌。動物實驗則將使用TLR4/CD14 基因剔除鼠模型。我們將使用細菌脂多醣體對動物模型刺激，以西方墨點法偵測脂多醣體刺激之後產生發炎的系列訊息傳導反應的相關分子，如MyD88, MAPK, 與 NF-kB 。也將以TUNEL assay 偵測TLR4/CD14 基因剔除鼠的腸道表皮細胞的凋亡。老鼠的糞便也將收集作聚合酶鏈鎖反應配合變性梯度膠電泳的方法予以分析。 我們預期本研究可以幫助了解基因、腸道內細菌交互作用，再造成腸道細胞的發炎或凋亡的步驟。我們可以發現克隆氏症的病人其腸內菌叢在疾病發作予緩解期不同的型態。這些結果將有助於解釋為何克隆氏症的發生率節節上升，以及未來治療的方向。<br> Abstract: Background and Aim: Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are common causes of chronic gastrointestinal disease in the developed world. CD is still very unfamiliar for the physicians here although its incidence of CD is increasing in Taiwan. The pathogenesis of IBDs is complex and both genetic and environmental factors contribute to its etiology. The first susceptibility gene, NOD2/CARD15 for CD, is strongly associated with European populations but its mutation is absent in Asian CD populations. Our previous study found ATG16L1 and TNFSF15 might be associated with adult CD in Taiwan. Due to small sample size, We need to further analyze the possible correlation between the ATG16L1 and TNFSF15 genotypes and clinical phenotype. The intestine flora might play a role in the initiation and perpetuation of the inflammatory process. However, there is no conclusion about the microbiota patterns in the initiation of IBD. To date, there is no compelling evidence of an etiological role for any single pathogenic micro- organism. The pathogenic or commensal bacteria may exert the inflammation through bacterial lipopolysaccharide (LPS), which may combine TLR4 and CD14 in the intestinal epithelial cells and trigger the downstream inflammatory signals. In CD, the mutated TLR4 may trigger an exaggerated inflammatory response. This research will unravel the importance of microflora pattern in the different disease status of CD and help understand the modulation of inflammation through the manipulating the microflora. The interaction of the genetic polymorphism and intestinal microflora are of particular interest in CD patients. We hypothesize that high dosage of commensal bacterial products may play a role in induction of excessive epithelial cell death leading to impairment of gut barrier function and CD develops subsequently. In this study, we aim to investigate whether luminal exposure to commensal microbiota/LPS induce enterocytic inflammation/apoptosis in genetically mutant animal models and CD patients. Materials and Methods: We will recruit adult and pediatric CD patients. The diagnosis of CD is based on the clinical, radiologic, endoscopic, and histologic examinations. Their blood samples will also be subjected to genetic analysis for SNP of ATG16L1, TNFSF15, and so on. Their stool samples will be collected at different disease status and subjected to polymerase chain reaction (PCR) plus denaturing gradient gel electrophoresis (DGGE) study to analyze the microflora pattern. The universal primers for 16s rDNA will be utilized to amplify the bacterial DNA and then subjected to DGGE to separate all the bacterial DNA fragments and finally read their sequences to identify the bacteria in the stool samples. For animal models, TLR4/CD14 knockout mice model will be investigated for the LPS stimuli and its subsequent signal pathway, including MyD88, MAPK, and NF-kB by western blots and proinflammatory responses. The mouse intestinal epithelium will be checked for apoptosis by TUNEL assay. The stool samples of the mice will also be subjected to DGGE study. Expected Results: We expected we may obtain the genetic polymorphism of the CD patients, and the genotype may correlate with the phenotype. The microflora patterns in CD patients will be incorporated with the genetic polymorphism results and the signaling pathway. We may understand the LPS, any dominant bacteria, and any cytokine gene polymorphism may play the crucial roles in the initiation and perpetuation of CD. These results may help unravel the pathogenesis of CD and pave the way for the future therapy of this disease.克隆氏症 腸道微生物菌叢 腸道細胞凋亡 TLR4Crohn‘s diseasemicrofloraintestinal epitheliumapoptosisTLR4|