臺灣博碩士論文加值系統

胃腸道在攝取和消化食物扮演重要角色，也因此它暴露於許多的食源性抗原和微生物群中的細菌抗原。同時，腸道包圍著大量的免疫細胞，以識別和攻擊外來抗原和微生物的威脅。腸道上皮細胞是胃腸道的關鍵成員，負責免疫細胞和腸腔微生物之間的相互作用，以抵抗病原體侵襲，並且對共生的微生物產生耐受性。免疫—上皮間的通訊失調，會使得腸內平衡狀態被破壞，並導致腸道的免疫性疾病。些許的真菌凝集素被發現具有免疫調節活性，亦稱為真菌免疫調節蛋白（FIP），它們能夠刺激免疫細胞，並增強細胞激素的產生，而且能夠抑制過敏反應和具有抗腫瘤活性。在本研究，先以口服餵食小鼠，收取腸道組織來評估FIP的作用。實驗結果顯示在腸繫膜淋巴結（MLN），調節型樹突細胞（rDC）的數量和rDC反應基因的表現受FIP調控。更進一步在骨髓衍生的rDC證明FIP在rDC的調節。為了研究FIP在腸道上皮的作用，以抗癌藥刺激腸上皮細胞（IEC），來測定FIP對生長曲線和傷口癒合的影響。結果顯示FIP具有保護作用，能使抗癌藥物刺激後的IEC回復。總結，FIP在腸道中的活性，透過調節rDC和IEC的功能，而有助於調控腸道的動態平衡。

　The gastrointestinal tract plays major role in taking and digesting food. Therefore, it exposed to a lot of food-borne antigens and bacterial antigens in the microbiota. Simultaneously, the gut surrounds a large quantity of the immune cells to identify and attack foreign antigens and microbial threats. The intestinal epithelium is key member of gastrointestinal tract that charged with the interaction between immune cells and luminal organisms to against pathogens invasion versus create tolerance of commensal microbes. The dysregulated immune-epithelial communication defect the intestinal homeostasis and cause immunopathology in gut. Some fungal lectins have been identified with immunomodulatory activity also called fungal immunomodulatory protein (FIP). They are capable of stimulating the proliferation of lymphocyte, monocytes and enhancing production of cytokines, and are competent of inhibiting allergic reactions and anti-tumor activities. In this study, their immunomodulatory effects were evaluated using orally administration to mice and harvest intestine tissues. The population of regulatory dendritic cells (rDC) in mesenteric lymph nodes (MLN) and the expression level of rDC-responsive genes are regulated by FIP. Using bone marrow derived rDC further proved specific regulation of FIP on rDC. To further investigate the role of FIP on intestinal epithelium, the growth curve and wound healing assay of intestinal epithelial cells (IEC) from anti-cancer drugs treatment were performed when FIP stimulation. The recovery of IEC from FIP treatment after anti-cancer drug stimulation proved that FIP have protective effect on intestinal epithelium.　In conclusion, immunomodulatory activities of fungal lectin at gut via regulating function of rDC and IEC with help to intestine homeostasis.

摘要……………………………………………………………………………………….…1
Abstract………………………………………………….…………………….……2
縮寫………………………………………………….……………...………….……3
一、研究背景…………………………………………………………...…………4
一、消化系統的功能……………………………….………………………...4
二、腸表皮細胞(intestinal epithelial cells, IECs)………….……………4
三、腸道中的免疫細胞……………………………………..………………..5
四、醛脫氫酶(aldehyde dehydrogenase : ALDH)……………….………6
五、菇類凝集素………………………………………………………………..7
六、抗癌藥物對腸道表皮的作用………………………………………….9
二、研究目的………………………………………………..…………...………10
三、實驗材料與方法…………………………………………………………11
三、一分子生物實驗…………………………………………………………..11
三、一 (一)核醣核酸抽取……………………………………………….11
三、一 (二)反轉錄反應……………………….…………………………..11
三、一 (三)聚合酶連鎖反應……………………………..………………12
三、一 (四)即時聚合酶連鎖反應……………………………..………..12
三、一 (五)蛋白質電泳…………………………………...………………12
三、一 (六)西方墨點法……………………….…………………………..13
三、二細胞生物實驗……………………………………………….………….14
三、二 (一)細胞培養…………………………………….....…..…………..14
三、二 (二)骨髓細胞分化成調節型樹突細胞……………………..….14
三、二 (三)流式細胞儀分析細胞表面抗原……………….…………..14
三、二 (四)流式細胞儀分析醛脫氫酶活性……….……….………….15
三、二 (五)腸表皮細胞癒合試驗…………………..……...…………….15
三、二 (六)酵素連結免疫試驗…………..……………........…………….15
三、三動物實驗………………………………………...………...…………….16
三、三 (一)小鼠口服餵食……………………………..………………….16
三、三 (二)眼窩採血……………………………...……………………….16
三、三 (三)皮下注射……………………………….......………………….16
三、三 (四)腹腔注射………………………………………...…………….17
三、三 (五)脾臟萃取……………………………………...……………….17
三、三 (六)骨髓細胞萃取……………………………...…...…………….17
三、三 (七)組織切片與脫蠟………………………………...……..…….17
三、三 (八)蘇木素-伊紅組織染色……………………………..……….18
三、三 (九)組織免疫化學染色……………………………..……..…….18
三、三 (十)組織免疫螢光染色………………………………....……….19
三、四統計分析方法…………………...….…………………...……..……….20
四、結果………………………………………………...…………………………20
一、小鼠組織醛脫氫酶表現的分析……………………………………..20
二、小鼠骨髓細胞分化的樹突細胞之醛脫氫酶表現量及活性的分析………………………...………………………………………………..……21
三、腸表皮細胞的細胞凋亡蛋白的分析……………………….………23
四、腸表皮細胞的自由基含量……………………………..……………..23
五、腸表皮細胞的移動能力分析………………………...………………24
五、討論……………………………………………………………...……………25
六、參考文獻………………………………………….…………………………27
七、圖表……………………………………………………...……………………30

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