臺灣博碩士論文加值系統

腸道與肺部之間的聯繫在免疫作用中的角色變得越來越重要。腸道菌不平衡可導致腸道外的疾病，比如肺部，腦部，免疫疾病，兒童發育等等。腸道通透性的增加將導致腸道病原體或抗原通過腸壁屏障進入全身循環，引發免疫反應，造成疾病。我們的實驗包含兩個部分，一部分是肺炎造成肺損傷的研究,另一部分是腸道屏蔽的研究。清楚兩者機轉後，未來，可以利用修復腸道屏蔽功能，來減少遠端腸道外的疾病或感染。

呼吸器相關肺炎(ventilator associated pneumonia; VAP)是在加護病房常見的院內感染。由綠膿桿菌造成的呼吸器相關肺炎具有高發病率跟死亡率。第一部分實驗是要檢驗由綠膿桿菌造成的呼吸器相關肺炎所產生的肺部損傷的相關機轉。我們先由鼻腔給予WT以及JNK1 -/-小鼠綠膿桿菌，再讓小鼠接受呼吸器使用。其次，將小鼠肺泡的巨噬細胞取出，用綠膿桿菌刺激後，將收集的懸浮液作分析並將懸浮液由鼻腔給予到WT以及JNK1 -/-小鼠，再給予呼吸器照護，以此來分析肺損傷的狀況。此外也使用分離自 WT、JNK1 -/-和IKK△mye小鼠的巨噬細胞進行綠膿桿菌的體外刺激後，收集上清液，做檢測。結果顯示，先給予綠膿桿菌或者巨噬細胞被綠膿桿菌刺激後的上清液後，再使用呼吸器，小鼠肺部的嗜中性白血球浸潤程度、肺泡沖洗液的發炎性細胞激素及發炎反應前期的細胞激素和亞硝酸含量皆明顯地增加。核因子活化B細胞κ輕鏈增強子與c-Jun氨基末端激酶的相關路徑也產生反應。而巨噬細胞經體外刺激後收集的上清液中，顯著增加的TNF-α可能在呼吸器相關性肺炎的發生扮演重要的角色。當給予綠膿桿菌後再連接呼吸器時，JNK1 -/-小鼠的肺部發炎及損傷均減緩。然而當IKK△mye小鼠或NK1 -/-小鼠的巨噬細胞受刺激後，IKK△mye小鼠的巨噬細胞懸浮液TNF-α含量則明顯下降，而JNK1 -/-小鼠的巨噬細胞體外刺激後的上清液的TNF-α的含量卻是增加的。由本實驗顯示：綠膿桿菌經由活化巨噬細胞的NF-κB增加TNF-α的生成，活化肺部組織內 JNK的訊息傳遞路徑，而增加小鼠呼吸器相關性肺炎的發生。

第二部分實驗，我們使用活體多光子顯微鏡觀察腸道屏蔽功能，並分析腸道通透性的變化。我們使用野生型小鼠和STZ誘導的糖尿病小鼠，比較兩組的腸道形態學變化和腸道通透性。我們的研究顯示糖尿病小鼠的腸道通透性增加，這與增加的杯狀細胞有關。絕大多數的滲漏是通過杯狀細胞（transcellular pathway）實現的，而非細胞間隙通路。透過這種杯狀細胞途徑，FITC從腸腔進入中心絨毛。腸道通透性的增加可以解釋為什麼糖尿病腸道屏障功能障礙容易發生遠處腸道外感染，如肺部疾病，肝膿腫和敗血症。

The connection between the intestine and the lungs is becoming more and more important in the immune role. Intestinal dysbiosis can cause diseases outside the intestinal tract. Increase in intestinal permeability will cause the intestinal pathogen or antigens pass through the gut barrier into the systemic circulation, triggering an immune response. Our experiments consisted of two parts, the first part was pneumonia-induced lung injury studies, and the other part was the study of intestinal permeability. We try to evaluate the mechanisms of both conditions. We can restore the intestinal barrier to prevent, treatment of the gut barrier dysfunctional related disease.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection among intensive care unit (ICU) patients. Pseudomonas aeruginosa (PA) is the most common multi-drug resistant Gram-negative pathogen and VAP caused by PA carries a high rate of morbidity and mortality. Our first part study examined the molecular mechanism of PA VAP-induced lung injury. C57BL/6 wild-type (WT) mice and JNK1 knockout (JNK1 -/-) mice received mechanical ventilation (MV) for 3 h at 2 days after receiving nasal instillation of PA. The WT and JNK1 -/- mice also received MV after the induction of lung injury by instillation of supernatants from PA-stimulated alveolar macrophages (AMs). AMs isolated from WT, IκB-kinase (IKK) βΔMye (IKKβ was selectively deleted in macrophages), and JNK1 -/- mice were ex vivo stimulated with live PA and supernatants were collected for cytokine assay. MV after PA instillation significantly increased the expression of ICAM and VCAM in the lungs and cytokines in bronchoalveolar lavage fluid (BALF) of WT mice, but not in JNK1 -/- mice. MV after supernatant instillation induced more total protein concentration in BALF and neutrophil sequestration in the lungs in WT mice than JNK1 -/- mice and cytokine assay of supernatants indicated that TNF-α is a critical regulator of PA VAP-induced lung injury. Ex vivo PA stimulation induced TNF-α production by AMs from WT as well as JNK1 -/- mice but not IKKβΔMye mice. In summary, PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. These results suggest that the pathogenesis mechanism of PA VAP involves production of TNF-α through activation of IKK/NF-κB pathways in AMs and JNK signaling pathway in the lungs.

In the second part study, we use intravital multi-photon microscopy to observe the intestinal function, and analyze the changes of intestinal permeability. Wild type mice and STZ induced diabetic mice were used. We compare the morphologic changes and intestinal permeability in both groups. Our study showed that increased intestinal permeability in diabetic mice was associated with increased goblet cells. Most of the leakage is achieved through the goblet cells (transcellular pathway) but note gap pathway. By this transcellular pathway, FITC enter the central villi from the intestinal lumen into lymphatic circulation and systemic circulation. This increase in intestinal permeability, can explain why gut barrier dysfunction in diabetes is prone to distant infection, such as lung disease, liver abscess and sepsis.

目錄------i
誌謝------ii
中文摘要------iii
English Abstract------v
圖目錄------vii
第一部 綠膿桿菌造成肺損傷之相關研究
第二部 多光子顯微鏡下，活體狀態探討腸屏蔽功能
引言------1
第一部分------1
第二部分------4
研究方法------6
第一部分------6
第二部分------10
結果------12
第一部分------12
第二部分------16
討論------18
第一部分------18
第二部分------19
介紹的圖------22
實驗結果圖------34
索引------122

圖目錄

介紹的圖Figure of introduction

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實驗結果圖

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