人體的腸道內有密集且多樣的微生物菌落，一般正常的腸道菌群包括大約400種不同的細菌種類，包含益生菌(probiotics)及致病菌，這些統稱為片(互?)利共生微生物菌叢(commensal microbiota)。Probiotics在人類營養和健康中具有重要的作用，例如避免致病菌的群聚及維持腸道免疫力。沙門氏菌是台灣夏季常見的病原菌，沙門氏菌感染症狀有噁心、嘔吐、腹痛、腹瀉等，嚴重可能導致死亡，在腸道則會造成淋巴耗損(lymphoid depletion)與壞死性腸炎(necrotizing ileitis)的發生。轉化生長因子β(transforming growth factor-β, TGF-β)磷酸化後經由活化Smad2/3/4蛋白質可增加IκBα(nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor)的生成量，來達到減少細胞核轉錄因子(Nuclear Factor-kappa B, NF-κB)、腫瘤壞死因子(tumor necrosis factor α, TNF-α)及介白素8(interleukin 8, IL-8)的表現，但在發炎時Smad7則會抑制此一路徑，增加NF-κB、TNF-α與IL-8的表現。本研究利用乳酸菌及沙門氏菌模式菌株為實驗材料，配合人類腸癌細胞株（human colon cancer cells, Caco-2）以TGF-β誘發保護之實驗模式，探討probiotics是否透過TGF-β訊息傳遞路徑調控Caco-2細胞之表現而降低沙門氏菌感染所造成的腸道發炎反應。由實驗結果可知，在不同濃度的沙門氏菌感染下，NF-κB在107菌落形成單位(colony-forming unit, CFU)/ml有顯著的活性表現(p<0.01)，而probiotics預處理的細胞在有沙門氏菌感染下，其TNF-α及IL-8的表現及NF-κB的活性明顯著地降低（p<0.05），同時，其TGF-β的表現卻明顯地增加（p<0.05）。IκBα在probiotics及益菌素(prebiotics)的預處理後感染組表現量則較僅有沙門氏菌感染組為高，而Smad7在沙門氏菌感染組表現量高於probiotics、prebiotics及合菌素(synbiotics)三組預處理後感染組。另一方面，微型核糖核酸microRNA-21(miR-21)可抑制Smad7蛋白質的合成，達到降低發炎的結果，本研究進一步以miR-21進行分析，觀察其對Smads家族及IL-8訊息路徑之調控作用。由試驗之結果可見，在益生菌的保護效果中，感染組的miR-21分泌量較probiotics及synbiotics預處理後感染組為少。因此，綜合上述之實驗結果發現益生菌能透過增加Caco-2細胞內的TGF-β/IκBα 及miR-21/Smad7的表現而降低NF-κB及IL-8的表現以減緩沙門氏菌所造成的腸道發炎反應。

Human intestine contains a dense and diverse community of microorganisms. Normal intestinal microflora comprises an estimated 400 different bacterial species, including probiotics and pathogens; these microbes collectively referred to as the commensal microflora, have an important role in human nutrition and health. Salmonella is a common pathogen during summer in Taiwan. The symptoms of salmonellosis include acute responses, such as nausea, vomiting, abdominal pain, diarrhea, even death, and chronic inflammatory response, such as lymphoid depletion and necrotizing ileitis in the intestine.
TGF-β (Transforming growth factor β)-phosphorizates and activates Smad2/3 to increase IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor), subsequently reduces inflammatory factors, such as NF-κB (Nuclear Factor-kappa B), TNF-α (tumor necrosis factor α) and IL-8 (interleukin 8) expression. Further, Smad 7 negatively regulates TGF-β signaling pathway to augment inflammation. In this study, probiotics (Lactobacillus acidophilus) and Salmonella, and human intestinal Caco-2 cells were employed to investigate how probiotics attenuates Salmonella-mediated intestinal inflammation. The results showed that Salmonella infection induced maximal NF-κB expression and TNF-α secretion at the concentrations of 107 CFU/ml; however, probiotics-pretreated cells had significantly lower TNF-α and NF-κB than those with Salmonella infection alone (p<0.05). They also had relatively higher activity of TGF-β/Samd3/4 (p<0.05). Moreover, IκBα expression in probiotics-pretreated cells was higher than that of S. typhimurium infection alone, but Smad 7 expression was lower in probiotics-pretreated cells. Consistent with Smad7 expression, miR-21, a down-regulator of Smad 7, was significantly higher in probiotics and synbiotics-pretreated cells compared with that of S. typhimurium infection alone. The experimental results showed that probiotics effectively attenuated Salmonella-induced intestinal inflammation in human intestinal Caco-2 cells via TGF-β1/Smads and TGF-β1/miR21 signaling pathway.

論文審定書1
誌謝3
中文摘要5
Abstract7
Abbreviation9
Introduction10
Materials and Methods13
Results18
Discussion21
Figures and Legend24
Appendix37
References45

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