臺灣博碩士論文加值系統

氣喘為慢性呼吸道發炎疾病，為免疫系統中TH1/ TH2類型細胞激素分泌不平衡所導致，其中一病理機制為TH2細胞所分泌之IL-5活化並吸引嗜酸性白血球浸潤至支氣管發炎部位，釋出更多的促發炎物質，加劇呼吸道發炎情形。雖然臨床上常使用類固醇藥物減緩發炎情況以改善病情，但是藥物的長期或不當使用往往伴隨副作用的產生。本實驗室先前研究發現卵白蛋白（ovalbumin, OVA）致敏之呼吸道發炎模式小鼠，餵予蜂膠當中的活性物質，咖啡酸苯乙酯（caffeic acid phenethyl ester, CAPE），連續五天（5, 10 or 20 mg/kg BW），可減少呼吸道發炎小鼠體內嗜酸性白血球浸潤至肺部發炎部位，增加脾臟中分泌IFN-γ細胞激素之細胞百分比，並且抑制脾臟細胞培養上清液中IL-5細胞激素分泌量，推測CAPE可能具有免疫調節之功效，而達到緩和呼吸道發炎小鼠之氣喘症狀。因此本論文欲以OVA致敏呼吸道發炎小鼠之研究模式，釐清CAPE改善呼吸道發炎之可能機制。實驗將呼吸道發炎小鼠於致敏後連續給予五天CAPE（20 mg/kg BW）或氣喘治療藥物prednisolone（5 mg/kg BW），發現CAPE可顯著降低呼吸道發炎小鼠之呼吸道阻力反應（airway hyperresponsiveness, AHR），且效果幾乎等同於藥物組，雖然CAPE也可顯著降低BALF中嗜酸性白血球之百分比，但是對於BALF中嗜酸性白血球實際細胞數、細胞激素IL-5分泌量、血清中總IgE和OVA特異性IgE生成量則無顯著影響。將呼吸道發炎小鼠純化出脾臟CD4+ T細胞，給予PMA-ionomycin刺激培養，結果發現餵予CAPE可顯著降低呼吸道發炎小鼠脾臟CD4+ T細胞於刺激下分泌IL-10和TGF-β1，而IL-5、IFN-γ分泌量與CD4+ T細胞增生反應和呼吸道發炎小鼠相比無顯著差異。若將呼吸道發炎小鼠脾臟CD4+ T細胞和CAPE（1, 2.5 or 5 μg/mL）進行體外培養，於PMA-ionomycin刺激下，隨著CPAE培養濃度愈高，CAPE對於細胞激素IL-5、IFN-γ和IL-10分泌量之抑制效果愈加顯著。為了進一步確認CAPE是否直接作用於嗜酸性白血球，將呼吸道發炎小鼠BALF細胞和不同濃度CAPE進行體外培養，在不影響細胞存活率和細胞數的前提下，CAPE可顯著降低嗜酸性白血球於BALF中所佔之百分比，但對於ConA刺激下之IL-5分泌量則無影響。綜合上述，呼吸道發炎小鼠於致敏後餵予CAPE有較佳之改善效果，推測CAPE直接或間接（CAPE代謝衍生物）藉由降低呼吸道發炎小鼠AHR反應、降低嗜酸性白血球浸潤至肺部或誘導嗜酸性白血球細胞凋亡，亦或者藉由調控脾臟CD4+ T細胞分泌IL-5，因而降低嗜酸性白血球趨化至肺部。所以CAPE或許具有輔佐氣喘治療藥物使用之能力，但是未來仍需更多的研究證明其調控機制。

Asthma is a chronic airway inflammation disease caused by the TH1/ TH2 cytokines secretion imbalance. One of the mechanisms is IL-5 secreted by TH2 cells activating eosinophils infiltration to airway inflammation site, and to release more pro-inflammatory mediators aggravating symptoms. In clinical, steroids are often used to improve inflammation, but long term or improper dependence on drugs would follow some complications. Previous study shown OVA (ovalbumin)-immunized airway inflammatory mice model fed with 5, 10 or 20 mg/kg BW caffeic acid phenethyl ester (CAPE), the active compound in propolis, for continuous 5 days. The CAPE could reduce eosinohilia in bronchoalveolar lavage fluids (BALF), increased the percentage of IFN-γ-secreting cell in splenocyte, and inhibited IL-5 secretion in splenocyte cultured supernatant. Therefore, we suggested CAPE has immunomodulatory activity on attenuating airway inflammatory mice. In this thesis, we would like to use OVA-immunize airway inflammatory mice model to clarify the possible mechanism on attenuating airway inflammation. We fed CAPE (20 mg/kg BW) or prednisolone (5 mg/kg BW), the asthma therapy medicine, to airway inflammatory mice after immunization for continuous five days. Our data shown that CAPE could significantly reduce airway hyperresponsiveness (AHR) to methacholine in airway inflammatory mice as low as the AHR of mice fed with prednisolone. Although the eosinophil percentage in BALF significantly suppressed by CAPE treatment, there had no effects on the eosinophil absolute numbers, IL-5 secretion, serum total IgE and OVA-specific IgE production. In order to understanding if CAPE regulated splenocyte CD4+ T cell to secret IL-5 that caused IL-5 reduction in BALF. We isolated CD4+ T cell from splenocyte in airway inflammatory mice fed with CAPE or prednisolone for continuous five days. After stimulating with PMA-ionomycin, the IL-10 and TGF-β1 secretion were significantly suppressed by CAPE. But the IL-5, IFN-γ secretion and CD4+ T cell proliferation had no effects compared to the asthma group. However, the ex vivo study shown that PMA-ionomycin-stimulated CD4+ T cell isolated from airway inflammatory mice incubated with different concentrations of CAPE (1, 2.5 or 5 μg/mL) suppressed the IL-5, IFN-γ and IL-10 secretion in a dose-dependent manner. In order to concern whether CAPE may affect on eosinophil directly, we incubated BALF cell collected from airway inflammatory mice with different concentration of CAPE ex vivo. Our data shown that CAPE could significantly reduce eosinophil percentage in BALF cells, but there had no effects on cell viability, numbers, and the secretion levels of IL-5 as cell been stimulated by Con A. In conclusion, according to our study design, the proper effect on improving airway inflammatory mice was fed with CAPE after OVA-immunization. We suggested that CAPE may attenuating airway inflammation on reducing AHR reaction, suppressing eosinophil infiltration to airway, inducing eosinophil apoptosis, or regulating splenocyte CD4+ T cell to secret IL-5, directly or indirectly (through some metabolic derivatives of CAPE). CAPE maybe used to an adjuvant for asthma therapy, however, there more studies are needed to demonstrate the regulatory mechanism in the future.

中文摘要............................................ I
英文摘要............................................ III

縮寫表.............................................. VI

第一章 前言............................................ 1
第二章 文獻回顧
第一節 呼吸道發炎反應
2.1.1 免疫反應......................................... 3
2.1.2 輔助型T細胞之調節................................. 3
2.1.3 發炎媒介物質...................................... 4
第二節 咖啡酸苯乙酯
2.2.1 咖啡酸苯乙酯簡介.................................. 5
2.2.2 咖啡酸苯乙酯之生物活性............................. 6
2.2.3 咖啡酸苯乙酯對T細胞調節之影響....................... 8
第三節 嗜酸性白血球
2.3.1 嗜酸性白血球在呼吸道發炎中扮演之角色................. 8
2.3.2 細胞激素對嗜酸性白血球之調節........................ 9
2.3.3 嗜酸性白血球與呼吸道重組........................... 10
2.3.4 抑制嗜酸性白血球活性之藥物.......................... 11
第四節 氣喘治療藥物
2.4.1 常見氣喘藥物...................................... 12
2.4.2 氣喘藥物之作用機制................................. 12
第三章 研究動機與目的.................................... 14
第四章 呼吸道發炎模式小鼠餵食CAPE對肺功能之影響
第一節 材料方法
4.1.1 試驗物質......................................... 16
4.1.2 建立呼吸道發炎模式小鼠............................. 17
4.1.3 實驗分組......................................... 18
4.1.4 呼吸道阻力值測試.................................. 19
4.1.5 血清OVA特異性IgE抗體之測定......................... 20
4.1.6 收集肺部沖洗液.................................... 21
4.1.7 Liu’s staining.................................. 21
4.1.8 細胞激素之測定.................................... 21
4.1.9 統計分析......................................... 22
第二節 結果
4.2.1 CAPE對呼吸道發炎小鼠呼吸道阻力值之影響............... 22
4.2.2 CAPE對呼吸道發炎小鼠肺部沖洗液嗜酸性白血球之影響..... 23
4.2.3 CAPE對呼吸道發炎小鼠肺部沖洗液細胞激素IL-5之影響..... 25
4.2.4 CAPE對呼吸道發炎小鼠血清OVA特異性IgE之影響.......... 26
第三節 討論............................................. 28
第五章 呼吸道發炎小鼠餵食CAPE對脾臟細胞分泌細胞激素之影響
第一節 材料方法
5.1.1 試驗物質......................................... 31
5.1.2 建立呼吸道發炎模式小鼠............................. 31
5.1.3 實驗分組......................................... 31
5.1.4 脾臟細胞之取得.................................... 32
5.1.5 純化CD4+ T細胞................................... 32
5.1.6 細胞表面抗原分析.................................. 33
5.1.7 活體外試驗部分.................................... 33
5.1.8 MTT assay....................................... 34
5.1.9 細胞激素測定...................................... 35
5.1.10 統計分析......................................... 35
第二節 結果
5.2.1 CAPE對呼吸道發炎小鼠CD4+ T脾臟細胞細胞增值之影響..... 35
5.2.2 CAPE對呼吸道發炎小鼠CD4+ T脾臟細胞於PMA-ionomycin
刺激下分泌細胞激素之影響........................... 37
5.2.3 純化自呼吸道發炎小鼠CD4+ T脾臟細胞與不同濃度CAPE
共同培養對PMA-ionomycin刺激下分泌細胞激素之影響..... 38
第三節 討論............................................. 42
第六章 CAPE對呼吸道發炎小鼠肺部嗜酸性白血球與細胞激素之影響
第一節 材料方法
6.1.1 試驗物質......................................... 44
6.1.2 建立呼吸道發炎模式小鼠............................. 45
6.1.3 收集肺部沖洗液.................................... 45
6.1.4 活體外培養........................................ 45
6.1.5 Liu’s staining..................................46
6.1.6 細胞激素測定...................................... 46
6.1.7 統計分析......................................... 46
第二節 結果
6.2.1 CAPE對肺部沖出細胞細胞存活率之影響.................. 46
6.2.2 CAPE與肺部沖出細胞共同培養後對各類細胞族群消長之影
響.............................................. 46
6.2.3 CAPE對肺部沖出細胞培養於Con A刺激下分泌IL-5之影響... 48
第三節 討論............................................. 51
第七章 總結............................................. 52
第八章 參考文獻......................................... 53

附錄一 常用試劑與配製方法................................. 64
附錄二 自人類週邊血中純化嗜酸性白血球...................... 66

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