臺灣博碩士論文加值系統

當過敏原進入人體組織時，樹突狀細胞會執行吞噬能力，將過敏原吞進細胞中進行抗原處理，並在淋巴組織中呈獻抗原給naïve T淋巴細胞進行T細胞的分化與增生，此外也能活化週邊組織中的作用T細胞 (effector T cell)產生反應。呼吸道的樹突狀細胞能吞噬過敏原，造成宿主對過敏原產生過敏，誘導Th2免疫反應產生，使得呼吸道充斥嗜酸性白血球並造成發炎，此外亦能造成Th2作用細胞活化，徵募至發炎部位執行作用。但目前仍不清楚塵蟎 Der p對樹突狀細胞的分化及功能的影響。Dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN, CD209) 為一種C-type lectin，透過carbohydrate recognition domain (CRD)能和具有mannose及fucose的醣化抗原做結合。先前在實驗室中發現到在台灣常見的過敏原塵蟎Dermatophagoides pteronyssinus (Der p) 能和DC-SIGN結合 (binding)。因此，我們假設當未成熟樹突狀細胞藉著DC-SIGN和塵蟎Der p結合作用後會影響樹突狀細胞的分化，並且在成熟階段的樹突狀細胞藉DC-SIGN和塵蟎Der p作用時會誘導Th2免疫反應的產生。於是，我們利用過敏病人以及非過敏正常人週邊血中單核球，以GM-CSF、IL-4刺激細胞生長分化成未成熟樹突狀細胞 (medium-MDDCs)；此外，在細胞激素刺激下，加入塵蟎 Der p共同培養，得到Der p-MDDCs，進行相關功能以及表現型的分析。在6-7天的分化過後，Der p-MDDCs表現較低比例的CD14-/CD1a+的樹突狀細胞，顯示Der p影響到樹突狀細胞的分化，但HLA-DR、CD80、CD86以及C-type lectin- MMR (macrophage mannose receptor) 卻與medium-MDDCs表現量並無差異性。然而在樹突狀細胞表面的DC-SIGN在Der p-MDDCs的表現量較medium-MDDCs來的低，並且過敏病人的DC-SIGN表現量也較正常人來的低。而在功能上的分析，首先於吞噬能力的觀察上，發現到Der p-MDDCs的吞噬能力較medium-MDDCs強，此外，過敏病人的吞噬能力也較正常人強。而於製造細胞激素的方面，未成熟樹突狀細胞以LPS刺激時會有大量的IL-10以及IL-12p40的分泌，但LPS結合Der p以及具刺激訊號能力的DC-SIGN單株抗體共同作用下和LPS相比較，會有IL-10分泌量增加以及IL-12p40分泌量減少的現象。進一步以自體的CD4+ T淋巴細胞和不同處理的樹突狀細胞共同培養，在LPS結合Der p、mannan以及 DC-SIGN抗體共同刺激的medium-MDDCs，相較於LPS刺激的細胞，有較強的能力誘導CD4+ T淋巴細胞表現GATA3 (Th2轉錄因子)。接下來探討Der p-MDDCs中DC-SIGN減少的原因，利用共軛焦顯微鏡實驗，可以觀察到FITC-Der p和未成熟樹突狀細胞共同培養，DC-SIGN會由細胞膜進入到細胞裡面，並和Der p有位置重疊的現象。這些實驗結果告訴我們，塵蟎 Der p和未成熟樹突狀細胞上的DC-SIGN結合作用下，會使得樹突狀細胞誘導走向Th2免疫反應。

Dendritic cells (DCs) are crucial in determining the outcome of allergen encounter and integrating signals derived from the antigen into a signal that can be read by naïve T cells in the lymphoid tissues and by effector T cells in peripheral tissues. Airway DCs play important roles not only in the sensitization to inhaled allergens but also in the maintenance of eosinophilic airway inflammation by inducing Th2 responses in the draining nodes and controlling the recruitment and activation of Th2 effector cells within sites of inflammation. But the detail event in the determination of DCs differentiation and function when contact with allergen is not clear. Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) is one of the important C-type lectins of dendritic cells which interacts with a large array of pathogens. Previously, we have found DC-SIGN could also bind allergen- Dermatophagoides pteronyssinus (Der p). In this study, we hypothesize that Der p allergen may interfere in the development of monocyte-derived immature DCs (MDDCs) and induce Th2 type of immune response in the maturation stage of DCs via interaction with DC-SIGN. To obtain MDDCs, PBMCs collected from Der p-sensitized individuals and non-atopic controls were subjected to 7-day incubated period in the presence of GM-CSF, IL-4, and with or without Der p allergen stimulation. After incubation, Der p-incubated MDDCs (Der p-MDDCs) were lower in the percentage of CD14-CD1a+ DCs, which suggested Der p effect DCs differentiation, but the expression of HLA-DR, CD80, CD86 as well as the other C-type lectin, MMR (macrophage mannose receptor) were the same as those in MDDCs without Der p incubation (medium-MDDCs). However, we found the cell surface expression level of DC-SIGN was significantly reduced in medium-MDDCs obtained from atopic patients as compared to those in non-atopic controls and MDDCs differentiation in the presence of Der p. The phagocytosis ability was higher in Der p-MDDCs than medium-MDDCs, and also in atopic patients than non-atopic controls. When immature MDDCs were stimulated with LPS to obtained matured DCs, there was decrease IL-12p40 and increase IL-10 cytokine productions from LPS-stimulated mature DCs combined with Der p or anti-DC-SIGN antibody. Autologus CD4+ T lymphocytes were co-cultured with immature MDDCs which were stimulated with LPS combined Der p, mannan or anti-DC-SIGN antibody produced more GATA-3 (Th2 transcription factor) than LPS alone. From confocal microscope examination we found the DC-SIGN and FITC labeled-Der p were colocalized in the MDDCs. These results suggested that Der p reacted DC-SIGN (CD209) of immature DCs may prone these cells to elicit Th2 immune response.

中文摘要 I
英文摘要 III
誌謝 V
總目錄 VI
圖表目錄 VIII

緒論 1
1.1 氣喘 1
1.2 塵蟎 1
1.3 致病機轉與症狀 2
1.4 樹突狀細胞與Th1、Th2免疫反應 3
1.5 氣喘與樹突狀細胞 4
1.6 DC-SIGN芝 5
1.7 過敏與DC-SIGN 7

研究目的與實驗設計 10

實驗材料與方法 13
2.1 藥品 13
2.2 抗體 14
2.3 耗材 15
2.4 儀器 15
2.5 血液樣本 16
2.6 分離週邊血單核細胞 16
2.7 純化單核球 17
2.8 體外分化樹突狀細胞 17
2.9 樹突狀細胞吞噬能力測定 18
2.10 酵素結合抗體測定法 (ELISA) 18
2.11 樹突狀細胞-T淋巴細胞共同培養 19
2.12 反轉錄聚合酶鏈鎖反應 (RT-PCR) 19
2.13 明膠蛋白酵素電泳法 (Gelatin zymography) 22
2.14 塵蟎Der p抗原接螢光 23
2.15 免疫螢光染色法 24
2.15.1 流式細胞儀 24
2.15.2 共軛焦顯微鏡 24
2.16 統計分析 25

結果 26
討論 32
圖表 38
參考文獻 55

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