臺灣博碩士論文加值系統

氣喘是一種第二型輔助T細胞( T helper cell )和嗜酸性球(eosinophil ) 媒介的慢性呼吸道發炎反應。近20年來，氣喘的罹患率和死亡率逐年提高。流行病學研究歸納指出，環境愈來愈乾淨，使得孩童們在幼兒時期失去接觸環境中一些微生物的機會，使體內Th1/Th2發展不平衡，可能是導致過敏性氣喘增多的原因。細菌性的感染，例如Mycobacteria，誘發Th1反應，有研究指出之與氣喘的發生有負相關；反之，呼吸道融合病毒( respiratory syncytial virus，RSV )的感染，則有助於氣喘的形成。
Dermatophagoides farinae ( Der f )是最常引起過敏性氣喘的家塵蟎，我們過去的研究發現，以多次氣管內接種Der f可致敏小鼠，並且引起肺部嗜酸性球發炎反應和Th2型細胞激素。本研究延續這個系統，事先給予牛結核桿菌( Bacillus Calmette-Guérin，BCG )感染的Der f致敏小鼠呼吸道嗜酸性球浸潤較輕微、肺泡沖洗液中IL-12、IFN-γ較高、TNF-α和IL-6較低、血中Der f特異性IgG1、IgG2a/2b較少，而且取出的肺部及淋巴結內分泌IFN-γ的T細胞比例較高。而先給予RSV，呼吸道嗜酸性球浸潤較嚴重、肺泡沖洗液中IL-12、IFN-γ較低、TNF-α和IL-6較高、血中Der f特異性IgG1、IgG2a/2b較多，取出分泌IL-4的T細胞比例高。由動物實驗結果得知BCG能減弱而RSV些微加重Der f的致敏和所引發的呼吸道發炎反應。
肺泡巨噬細胞( alveolar macrophage )是肺部非常重要的防禦細胞，因此推測在感染與氣喘的形成過程中可能扮演一個重要的角色。由感染BCG或RSV小鼠中分離出肺泡巨噬細胞對Der f刺激有不同的反應，BCG感染的肺泡巨噬細胞對Der f刺激只釋放少量IL-6，且BCG會干擾Der f對肺泡巨噬細胞的輔助作用的影響；反之，RSV感染的肺泡巨噬細胞本身產生IL-6，在Der f刺激後IL-6產量更高。於In-vitro實驗中發現BCG無法抑制高濃度Der f刺激肺泡巨噬細胞IL-6的釋放，但可以抑制較低濃度Der f刺激肺泡巨噬細胞IL-6的釋放，而RSV不但本身會引起肺泡巨噬細胞釋放IL-6且加入Der f更有加成的作用。因此我們推論肺泡巨噬細胞在BCG與RSV感染後有不同的反應，特別在IL-6的釋放上，造成BCG減弱而RSV加劇由Der f引發致敏性和呼吸道發炎反應。

Asthma is a chronic inflammatory disease of the bronchial airways orchestrated by the type 2 helper T cells, eosiniphils and their secreted cytokines. The prevalence of asthma in modern, highly industrialized countries has been risen during the past two decades. It is argued that in a very clean environment without normal colonization pattern or infections in infancy may disturb Th1/Th2 balance. Bacterial infections, such as Mycobacteria, induce Th1 type response and prevent the progression of asthma. On the contrary, viral infections, such as respiratory syncytial virus (RSV), augment the symptom of bronchial inflammation.
Dermatophagoides farinae (Der f) is one of the most prominent and important species of house dust mite implicated in allergic asthma. In our previous study, we demonstrated that Der f was proinflammatory in mice, and that repetitive intratracheal challenge of Der f induced an allergic airway inflammation characterized by the infiltration of eosinophils and lymphocytes and elevation of IgE antibody and Th2 cytokine levels. Using this model system, we observed that pre-infection of mice with Bacillus Calmette-Guérin (BCG) significantly attenuated the Der f-induced eosinophilia in blood and BAL fluids, TNF-α and IL-6 levels in BAL fluids, and Der f-specific IgG1 and IgG2a/2b in serum as compared with non-infected mice. Intracellular cytokine staining of lung cells and lymph node cells revealed there were more IFN-g-positive cells and less IL-4-positive cells in the BCG-treated Der f-challenged mice. On the contrary, pre-infection with RSV deteriorated the Der f-induced airway inflammations.
Alveolar macrophages (AMs) are targets of mycobacteria, RSV and Der f. Therefore, we further examine how there pathogens affected the accessory function and mediator production of AMs in response to Der f. First, we observed that AMs from RSV-infected but not BCG-infected mice elaborated IL-6 ex-vivo. In addition, RSV-primed AMs produced substantial amounts of IL-6 after Der f stimulation. In contrast, BCG-primed AMs did not produce IL-6 in response to low concentration of Der f. In conclusion, this investigation demonstrates that (1)mycobacterial infections have the potential to suppress and RSV to augment the development of atopic disorder, (2)differences exist between BCG and RSV infection, particularly at IL-6 production of AMs.

目 錄
中文摘要……………………………………………………………………I
英文摘要……………………………………………………………………II
目錄…………………………………………………………………………III
圖表目錄……………………………………………………………………IV
第一章 緒論………………………………………………………………1
第一節 氣喘現況………………………………………………………1
第二節 過敏性氣喘致病機轉…………………………………………1
第三節 家塵蟎過敏原及其致敏性……………………………………2
第四節 環境因素與氣喘發生的相關性………………………………3
第五節 結核分枝桿菌( Mycobacteria )及氣喘的關係……………6
第六節 呼吸道融合病毒( Respiratory syncytial virus )感染及氣 喘的關係……………………………………………………………………7
第七節 肺泡巨噬細胞( Alveolar macrophages )與氣喘的致病機轉……………………………………………………………………………8
第八節 研究動機………………………………………………………9
第二章 實驗設計…………………………………………………………11
第一節 整體實驗構想…………………………………………………11
第二節 具體實驗目標…………………………………………………11
第三節 動物實驗的實驗設計…………………………………………12
第四節 Ex-vivo實驗的實驗設計………………………………………13
第五節 體外實驗的實驗設計…………………………………………14
第三章 材料與方法………………………………………………………15
第一節 方法……………………………………………………………15
A、萃取家塵蟎抗原 ( Dermatophagoids farinae )………………15
B、培養HEp-2細胞……………………………………………………16
C、培養呼吸道融合病毒 ( Respiratory syncytial virus，RSV )………………………………………………………………………………16
D、測定病毒力價………………………………………………………17
E、牛型分枝桿菌( Mycobacteria bovis, BCG )的使用…………18
F、實驗動物……………………………………………………………18
G、氣管內接種 ( Intratracheal inoculation )…………………18
H、支氣管肺泡沖 ( Bronchoalveolar lavage，BAL )……………19
I、支氣管肺泡沖洗液中白血球分類計算……………………………20
J、血中嗜酸性球的計數………………………………………………20
K、測定肺泡沖洗液或巨噬細胞刺激後上清液中IL-6、IL-12、TNF-α、IFN-γ和IL-4的含量……………………………………………………20
L、測定血清中全部IgE抗體和家塵蟎特異性IgG1、IgG2a抗體含量……………………………………………………………………………21
M、免疫螢光染色細胞內部細胞激素( Intracellular cytokine staining )…………………………………………………………………22
N、免疫螢光染色細胞表面抗原……………………………………………………………………………23
O、肺部巨噬細胞以家塵蟎、牛型分枝桿菌或呼吸道融合病毒刺激後的活性實驗…………………………………………………………………23
P、肺部巨噬細胞以家塵蟎、牛型分枝桿菌或呼吸道融合病毒刺激實驗……………………………………………………………………………24
Q、肺泡巨噬細胞影響T細胞的分化…………………………………25
R、肺泡巨噬細胞影響T細胞的增生…………………………………26
第二節 材料………………………………………………………………27
A、儀器設備……………………………………………………………27
B、藥品…………………………………………………………………28
第四章 實驗結果…………………………………………………………32
第五章 討論………………………………………………………………39
參考文獻……………………………………………………………………44
圖表…………………………………………………………………………49
附錄一 曾嘗試過的實驗…………………………………………………62
附錄二 有關BCG和RSV對氣喘模式影響的文獻中的實驗設計…………66
圖 表 目 錄
Fig. 1 BCG及RSV感染刺激小鼠後引起肺部發炎反應……………………49
Fig. 2 BCG感染小鼠後肺部的病理變化……………………………………50
Fig. 3流體細胞分析儀分析BCG和RSV感染後小鼠肺泡巨噬細胞B7.1、B7.2、CD1d的表現……………………………………………………………51
Fig. 4 BCG減弱而RSV加劇由Der f引發的呼吸道發炎……………………52
Fig. 5 BCG減弱而RSV增加Der f致敏小鼠血中嗜酸性球的數量…………53
Fig. 6 BCG減弱而RSV增強由Der f致敏小鼠血清中塵蟎特異性IgG1和IgG2a/2b 抗體的含量………………………………………………………54
Fig. 7 Der f、BCG、RSV刺激後小鼠肺泡沖洗液中IL-12、TNF-α、IL-6的含量…………………………………………………………………………55
Fig. 8 在Der f致敏小鼠感染BCG增加而RSV減少IFN-γ產生細胞/ IL-4產生細胞…………………………………………………………………………56
Fig. 9 從BCG或RSV感染小鼠分離的肺泡巨噬細胞對Der f刺激後釋放IL-6的差異…………………………………………………………………………58
Fig. 10 Der f、BCG、RSV對肺泡巨噬細胞釋放細胞激素的影響………59
Fig. 11 BCG減弱而RSV促進Der f誘發肺泡巨噬細胞IL-6的釋放………60
Fig. 12 BCG回復經Der f作用肺泡巨噬細胞後所喪失抑制T細胞增生的作用………………………………………………………………………………61

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