臺灣博碩士論文加值系統

過敏性氣喘是由Th2細胞所主導的慢性肺部發炎疾病。先前的研究顯示，地黃透過抑制FcεRI介導的信號傳遞途徑減少調控肥大細胞去顆粒化的程度以及前發炎細胞因子的分泌進而抑制過敏反應。地黃是一種用於治療哮喘的中草藥，而梓醇是一種從地黃的根分離出來的環烯醚萜苷。然而，梓醇對氣喘的抗過敏作用以及其潛在機制仍不清楚。因此，我們探討了梓醇對樹突狀細胞，T細胞和肥大細胞的免疫調節作用。 此外，我們也在OVA誘導的氣喘動物模式中探討梓醇治療是否可以抑制過敏性氣喘誘導的Th2反應。體外實驗的結果顯示，骨髓衍生的樹突狀細胞在使用梓醇刺激後會分泌較少的前發炎細胞因子(如IL-1β，IL-6和腫瘤壞死因子TNF-α)，並且也會使樹突狀細胞的細胞表面分子MHCⅡ表達量輕微降低。藉由混合淋巴球反應，可以得知經梓醇刺激的樹突狀細胞可以輕微抑制CD4+T細胞增生反應以及Th1的趨向。此外，梓醇直接抑制由anti-CD3 /anti-CD28抗體活化的CD4+T細胞的增生反應和細胞因子的產生（IL-5，IL-10，IL-13，IFN-γ)。梓醇作用在肥大細胞上，可以抑制OVA-IgE誘導的去顆粒作用跟介導物質的釋放（IL-6，TNF-α和IL-13）。然而，梓醇的刺激對肥大細胞表面分子FcεRI的表達沒有影響。在動物實驗中，口服梓醇可降低血清中OVA專一性的IgE的產生。也可以改善了呼吸道過度反應、杯狀細胞增生和發炎細胞浸潤在肺部的情況。同時，梓醇的治療可以抑制肺泡沖洗液和脾臟細胞培養液中，eotaxin、KC、Th2型細胞因子（IL-4，IL-5，IL-13）的產生。總而言之，這些研究結果的發現顯示T細胞和肥大細胞有潛力作為治療氣喘的標靶細胞。在這個研究中，我們為梓醇的抗發炎作用提供了新的證據，這有助於釐清梓醇做為治療Th2介導的過敏性發炎症的藥物評估。

Atopic asthma is a chronic inflammatory disease of the lungs that is a kind of Th2-associated asthma. Previous studies have shown that Rehmannia glutinosa suppresses allergic reactions by inhibiting the mast cells degranulation and inflammatory cytokine secretion via down-regulation of the FcεRI-mediated signaling pathway. Rehmannia glutinosa is a traditional Chinese herbal medicine used to treat asthma, and catalpol is an iridoid glycoside which is isolated from the fresh root of Rehmannia glutinosa. However, the anti-allergic effects of catalpol on asthma and the underlying mechanism is still unclear. Thus, the immunomodulatory effects of catalpol on dendritic cells (DCs), T cell and mast cells were explored. Furthermore, we examined whether catalpol treatment can suppress allergic Th2 respones in an ovalbumin (OVA)-induced asthma model. In vitro analyzes revealed that catalpol-treated bone marrow-derived DCs secreted low levels of proinflammatory cytokine such as IL-1β, IL-6 and tumor necrosis factor TNF-α and displayed slightly reduced levels of surface molecules of MHC class II. By mixed leukocyte reaction assay, we found that catalpol-treated DCs slightly inhibited CD4+T cell proliferation and Th1 polarization. Additionally, catalpol directly suppressed anti-CD3/anti-CD28 antibodies-activated CD4+T cell proliferation and cytokine production (IL-5, IL-10, IL-13, IFN-γ). Catalpol also inhibited the OVA-IgE-induced degranulation and the release of mediators (IL-6, TNF-α and IL-13) by mast cells. However, catalpol treatment had no effect on the expression of FcεRI by mast cells. In animal experiments, oral administration of catalpol reduce OVA-specific IgE production in serum. It also ameliorated thedevelopment of airway hyperresponsiveness, goblet cell hyperplasia and inflammatory cells infiltration in the lung. Meanwhile, catalpol treatment inhibited the production of eotaxin, KC, Th2-type cytokine (IL-4, IL-5, IL-13) in brochoalveolar lavage fluid and culture supernatant of spleen cells. Collectively, these findings suggest that T cells and mast cells are potential target cells responsible for the action of catalpol against allergic asthma. Here in, we provide new evidence for an anti-inflammatory role of catalpol, which facilitates the potential use of catalpol as an immunomodulatory adjuvant to treat Th2-mediated allergic inflammation.

誌謝...............................................................4
中文摘要...........................................................5
英文摘要...........................................................6
第一章 緒論
1. 氣喘疾病的簡介..................................................9
2. 氣喘疾病的致病機轉..............................................9
3. 樹突狀細胞在過敏性氣喘中扮演的角色..............................10
4. T細胞在過敏性氣喘中扮演的角色..................................10
5. 肥大細胞在過敏性氣喘中扮演的角色................................11
6. 氣喘的治療方法..................................................11
7. 梓醇的簡介......................................................12
第二章 探討梓醇對各種免疫細胞之成熟與活化的影響
研究動機與目的.....................................................16
研究材料與方法
1. 梓醇來源及處理..................................................18
2. 培養骨髓衍生之樹突狀細胞........................................18
3. CD4+ T細胞的純化................................................19
4.肥大細胞的培養...................................................19
5.細胞之存活率試驗.................................................19
6.細胞激素之檢測...................................................20
7.細胞表面分子之檢測...............................................21
8.T細胞增生之檢測.................................................22
9.去顆粒化的檢測...................................................22
10.西方墨點法......................................................23
11. 統計方法.......................................................23
研究結果...........................................................24
第三章 探討梓醇在氣喘動物模式中的治療效果
研究動機與目的.....................................................30
研究材料與方法
1. 過敏氣喘動物模式建立............................................31
2. 血清中OVA專一性抗體之測量.....................................32
3. 呼吸道阻力測試..................................................32
4. 肺部沖洗液中血球分類計算........................................32
5. 細胞激素之測量..................................................33
6. 肺組織切片染色..................................................33
7. 脾臟細胞增生反應................................................34
8. 脾臟細胞之細胞凋亡試驗..........................................34
研究結果...........................................................35
第四章 討論........................................................39
第五章 結論與未來方向..............................................44
圖表...............................................................46
參考文獻...........................................................76

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