過敏性氣喘(Allergic asthma)是一種慢性氣管發炎疾病，過敏肇因於針對過敏原的Th2淋巴球免疫反應失調，造成嗜酸性球、肥大細胞、上皮細胞的活化與增生，並增加氣管過度反應及黏液的增加。Dermatophagoides farinae (Der f)是造成過敏性氣喘的主要的室內塵蟎過敏原之一，塵蟎過敏原的致敏分子機制有許多未解的課題。許多文獻顯示後天性免疫系統深受先天性免疫系統所調控及影響並且在防禦系統上扮演一個很重要的角色。因此本論文第一部份是利用多次氣管內接種Der f，激發小鼠呼吸道發炎反應來探討塵蟎致敏小鼠過程中先天免疫細胞，如肺泡巨噬細胞(alveolar macrophages)、肥大細胞(mast cells)之角色。結果顯示，在活體內，Der f在非IgE抗體依賴下，使肥大細胞進行去顆粒作用，而此作用可經肥大細胞穩定劑 ( sodium cromoglycate, 40 mg/kg )有效地來抑制。多次Der f刺激則引發呼吸道嗜酸性球發炎及產生過敏原特異性抗體，肺泡沖洗液(bronchoalveolar lavage fluid；BALF)一氧化氮含量增高，為正常小鼠約15-25倍，但抗氧化物的產量卻降低50-75%。接種Der f之小鼠的肺泡巨噬細胞可以增加T細胞的增殖作用並伴隨B7分子的表現增加。此外，呼吸道融合病毒感染會增強經Der f所引發的呼吸道發炎及致敏。體外實驗顯示Der f可以活化NFB並促使肺泡巨噬細胞釋放發炎前趨物質如IL-6、TNF及一氧化氮，並有效抑制抗氧化物的產生。經Der f刺激的肺泡巨噬細胞會增加T細胞的增生作用，及促使Th2細胞的發展(有較高的IL-4的表現)。此外，肥大細胞與肺泡巨噬細胞在Der f刺激後會有交互作用，包括增加肺泡巨噬細胞B7輔助分子之表現、趨化基因之表現、也吸引T淋巴球及單核球，並促使Th2細胞的發展及增生。抑制性實驗證明B7.2分子、一氧化氮、抗氧化物等分子皆參與Der f刺激的肺泡巨噬細胞增強T細胞的增殖作用及炎性細胞激素的釋放。Lectin-like醣類分子及RGD peptides可有效競爭阻斷Der f對肺泡巨噬細胞之作用。此外，在IgE不依賴機制下，Der f可促使肥大細胞株(P815)及骨髓肥大細胞(bone marrow-derived mast cells) 釋放大量炎前趨細胞激素及表現第二型細胞激素基因。因此在我們認為肥大細胞及肺泡巨噬細胞致敏過程中扮演多種重要的角色，藉由釋放的發炎媒介物質來起始及放大呼吸道黏膜致敏及發炎反應。
　　論文第二部份則以二維電泳(two-dimensional gel electrophoresis，2-DE)及串聯式質譜儀(Q-TOF/MS)分析探討塵蟎誘發呼吸道發炎之肺泡沖洗液蛋白質的變化和身份，以提供致敏過程之診斷或治療依據。二維電泳結果發現單次給予Der f的蛋白質點多座落在pI 5.5~6.5之間，分子量則分佈在15~25 kDa。其所呈現的蛋白質表現型態明顯不同於LPS (lipopolysaccharides)及OVA (ovalbumin)的刺激，經統計分析，在Der f約有81.5%，LPS約有89.6%，OVA約有97.4%的蛋白質表現是重複出現的。隨著氣管給予Der f的次數增加，相對於單次Der f之刺激，低分子量(15~25 kDa)的蛋白質點的數量明顯降低。另一方面，在parental-sensitization模式，增加的蛋白質點大多座落在pI 5.4~8.3。接著，我們藉質譜儀鑑定二維電泳膠片上＜50 kDa的蛋白質，結果顯示單次Der f刺激小鼠所誘發的蛋白質點包含了急性期蛋白質、宿主防禦蛋白質、細胞骨架蛋白質及其他類蛋白質，酵素則包括了抗氧化物；在多次Der f誘發之慢性發炎的狀態下，肺泡沖洗液中除包含急性期蛋白，宿主防禦性蛋白質外，尚有掌控氧化及抗氧化平衡的酵素。surfactant-associate protein A、補體和誘發性抗體。在parental-sensitization模式下，肺泡沖洗液中有大量炎性分子如補體、ferritin、annexin A3、4。此外，chloride intracellular channel 3、cytokine、chemokine ligand及translational proteins、tyrosine 3/tyrosine 5-monoxygenase也大量表現。致敏的同時，給予抗氧化物N-acetylcystine (NAC)及肥大細胞穩定劑(SCG) 可有效減低細胞浸潤情況及其所誘發的蛋白質點。由上述結果得知，多次塵蟎刺激會使肺部趨向氧化及抗氧化平衡的反應，若經塵蟎致敏後再刺激則引發較嚴重的發炎，此時伴隨cytokine及chemokine的出現，這些蛋白質與過敏性氣喘之致敏機制的關係值得進一步研究探討。

　　Allergic asthma is a chronic airway inflammation, which is characterized by the infiltration of Th2 cells and eosinophils in the airways, airway hyperresponsiveness, and excessive mucus secretion. Dermatophagoides farinae (Der f) is one of the most prominent and important species of house dust mite implicated in allergic asthma. However, the allergenicity of Der f is not fully known. The first part of the thesis was to investigate whether the effect of Der f on innate immunity involved in allergic sensitization in a murine model of asthma with emphasis on alveolar macrophages (AMs) and mast cells (MC). In vivo results showed that Der f induced eosinophilic airway inflammation and allergen-specific antibody in mice after repetitive challenge. Administration of sodium cromoglycate, a mast cell stabilizer not only suppressed acute mast cell activation as revealed by the release of mMCP-1 but also attenuated the allergic airway inflammation. There was a 15- to 25-fold increase in nitric oxide (NO) and a 50–75% decrease in antioxidant concentrations in bronchoalveolar lavage fluids (BALF) of Der f mice. AM from Der f-challenged mice expressed enhanced levels of costimulatory molecule B7 and augmented T cell proliferation ex vivo. Furthermore, dexamethasone attenuated and live RSV infection augmented airway inflammation and sensitization in mice repetitive challenge with Der f. In vitro studies showed that Der f activated NF-B of AM and, unlike ovalbumin (OVA) or lipopolysaccharides (LPS) stimulation, it up-regulated IL-6, TNF-, and NO. In addition, Der f down-regulated antioxidants. Der f-stimulated AM expressed enhanced levels of B7 molecules, supported T cell proliferation, and promoted Th2 cell development. Arg-Gly-Glu-Ser (RGDS) peptide and neo-glycoproteins blocked Der f effects on AM. Der f but not OVA could rapidly induced an enhanced expression of the genes for IL-1, IL-4, IL-6, IL-9, IL-13 and TNF- in mystocytoma P815 cells, and stimulated P815 cells and bone marrow-derived mast cells (BMMC) to produce IL-4, IL-6 and TNF- in a dose- and time-dependent manner. Cycloheximide blocked the Der f-induced IL-4 production, indicating a de novo protein synthesis process. Furthermore, supernatants from Der f-stimulated mast cells not only chemoattracted monocytes and T lymphocytes but also up-regulated the expression of B7.1 molecule, eotaxin, RANTES, MCP-1, and IP-10 mRNA of AM; they supported PHA-induced T cells proliferation and promoted Th2 cell development. These results showed that AM and mast cells in the airway mucosa might facilitate the initiation and development of allergic sensitization and inflammation.
　　The second part of this thesis was intended to identify, quantify and characterize the BALF proteins related to the Der f-induced allergic airway inflammation using two-dimensional electrophoresis (2-DE) and mass spectrometer. Twenty four hours after a single Der f challenge, there was a significant increase in the number of silver-stained spots mostly in an area of the electropherogram between pI 5-7 and Mr <25 kDa. The intensity, and, in a lesser extent, the number of protein spots were gradually increased and sustained for at least 48 h, which was correlated with a cellular influx into the BALF. Der f-, OVA-, and LPS-challenged mice had a total of 276, 231, and 251 spots detected, and among them 82, 56 and 37 spots had a twofold increase in abundance, respectively. Image analysis showed that the majority of the spots in the three treatments were overlapped (82% of Der f, 97% of OVA and 90% of LPS). There was a subset of the induced spots specific to each allergen, 24 spots for Der f, 9 for OVA and 10 for LPS. Fifteen proteins were identified in the BALF samples of single Der f-challenged mice by mass fingerprinting. They were proteins involved in acute phase response, host defense, antioxidant defense, cellular structure, and other purpose. In the BALF of repetitive challenged mice, 224 spots were detected. Most of which clustered in an area of the electropherogram between pI 5-7 and Mr 25-50 kDa. Comparison of the BALF protein maps of the two groups showed 56 spots with significant variations in relative abundance. Among them 28 spots with greater than twofold increase in intensities were subjected to MS, and they were proteins related to inflammation, host defense, enzymes involved in oxidant and antioxidant balance, cytoskeleton and antibody. Collectively, this study provides a large-scale profile of protein expression in the lung of Der f-induced inflammation in mice. 2-DE/MS analysis in combination with cellular immunoassays will give a more deep insight into the disease mechanisms of allergic asthma.

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