臺灣博碩士論文加值系統

近來國人飲食習慣與生活環境改變，導致過敏性疾病如氣喘之罹患率逐年增加。先前研究發現餵食蜂膠組小鼠的呼吸道阻力值較控制組低，其脾臟細胞分泌之IFN- (interferon-) 較控制組增加，而IL-10 (interleukin-10) 分泌量則降低，推測蜂膠可能具改善氣喘之功能。為探討蜂膠成份改善呼吸道發炎疾病之可能機制，以蜂膠中之有效成份咖啡酸苯乙酯(caffeic acid phenethyl ester; CAPE) 為研究材料，利用卵白蛋白 (ovalbumin; OVA) 致敏BALB/c雌鼠產生過敏反應，再以吸入OVA誘發小鼠呼吸道發炎症狀。犧牲小鼠後，將腹腔抽出細胞及脾臟細胞與CAPE共同培養，探討其對脾臟細胞增生與細胞培養上清液中細胞激素的影響。另以轉殖IFN-質體之人類Jurkat T細胞株與CAPE共同培養，測定CAPE對細胞激素IFN-基因表現之影響。結果顯示，體外培養系統中，脾臟細胞在有絲分裂劑刺激下，包括細胞增生反應、IFN-與IL-4細胞激素分泌量均受CAPE抑制，Jurkat T細胞之IFN-基因表現亦受CAPE抑制，CAPE對於以LPS (lipopolysaccharides) 刺激腹腔抽出細胞培養上清液中IL-6與TNF- (tumor necrosis factor-) 有促進分泌之趨勢。此結果與先前研究活體動物餵食蜂膠之免疫反應不ㄧ致，故另外設計探討CAPE是否具調節呼吸道發炎小鼠功效之試驗。活體試驗中，將呼吸道發炎小鼠分為控制組、CAPE組 (每公斤小鼠每日管餵5、10或20毫克) 及藥物治療組 (每日管餵0.1毫克prednisolone)，再另設置未經致敏的non-treat組，共分為六組，管餵五天後犧牲小鼠，分析肺部沖洗液中嗜酸性白血球數目與發炎相關蛋白質含量，測定淋巴細胞增生情形，並分析腹腔細胞與脾臟細胞培養上清液中細胞激素分泌量。實驗結果發現，餵食CAPE組小鼠肺部沖洗液中嗜酸性白血球數目顯著較控制組低，以Con A (concanavalin A) 刺激脾臟細胞培養上清液中，IL-5細胞激素亦顯著低於控制組，餵食20毫克CAPE組小鼠脾臟中，分泌IFN-細胞激素的細胞百分比顯著高於控制組，CAPE亦促進T與B細胞增生，且餵食10或20毫克CAPE組小鼠腹腔細胞培養上清液中，IL-6與TNF-促發炎細胞激素分泌量有抑制情形。推測CAPE可能藉由減少IL-5細胞激素分泌，而抑制嗜酸性白血球趨化聚集至肺部氣管，透過其抗發炎作用而有助於改善呼吸道發炎症狀，研究結果將可作為開發緩解氣喘之佐劑的參考。

The dietary pattern and environmental status has changed, which in term causing an increasing rate of allergic disease such as asthma. Previous study showed that the ovalbumin (OVA)-sensitized mice fed with propolis shown lower air hyperresponsiveness, and the Interferon- (IFN-) secreted from splenocytes of propolis-fed groups was higher and interleukin-10 (IL-10) was lower than control group. According to this, we proposed that propolis has beneficial effects on asthma. The aim of the study will focus on finding the mechanisms of the compounds in propolis on OVA-sensitized murine model of airway inflammation. Firstly, we selected caffeic acid phenethyl ester (CAPE), an active component of propolis, as target material for elucidate the mechanism. BALB/c female mice were immunized and sensitized exposing to OVA antigen to establish the asthma animal model. The splenocytes and peritoneal exudated cells isolated from BALB/c mice were incubated with different concentration CAPE. The effects of CAPE on splenocytes proliferation and cytokine secretion profiles have been done. Additionally, gene transfected plasmid Jurkat cells were incubated with CAPE, and then the IFN- gene expression by DLR assay was to be concerned. Our data showed that the proliferative response, IFN- and IL-4 cytokines secretion of splenocytes which been stimulated with mitogen were inhibited when cocultured with CAPE. The IFN- gene expression on Jurkat cells was inhibited by CAPE. However, CAPE could stimulate IL-6 and tumor necrosis factor- (TNF-) secretion from peritoneal exudated cells. As previous described in vitro model didn’t give the reproducible results when compare to the pilot study of in vivo asthma animal model. So we developed an airway inflammation model for further evaluated the therapeutic roles on CAPE. In vivo study, airway inflammation model in BALB/c mice were divided into six groups including control group, CAPE group (5, 10 or 20 mg/kg BW/day), prednisolone group (0.1 mg/day), and non-treat group. Mice were sacrified after administered with above treatments for five days by tube feeding or not. The results showed that eosinophilia in bronchoalveolar lavage fluid (BALF) of the asthma animal model receiving CAPE were significantly lower than control group, and IL-5 secretion of splenocytse stimulated with Con A (concanavalin A) had the same trend. The results of intracellular staining demonstrated that the percentage of IFN- positive splenocytes from CAPE-administered mice (20 mg/kg BW/day CAPE group) were significant higher than control group. The proliferative response of T cells and B cells was increased by CAPE. Mice receiving CAPE (10 or 20 mg/kg BW/day) shown decreased secretion of the pro-inflammatory cytokines, such as IL-6 and TNF- compare to control group. According to these finding, we propose CAPE may inhibit the recruitment of eosinophils to airways by decreasing IL-5 cytokine secretion. The anti-inflammatory effect of CAPE will be helpful on airway inflammation. The study may provide novel applicable therapic effect on asthma.

中文摘要………………………………………………………………………………. I
英文摘要……………………………………………………………………………... III
目錄…………………………………………………………………………………….V
圖目錄……………………………………………………………………………….VIII
縮寫表………………………………………………………………………………….X
第一章 前言………………………………………………………………………… 1
第二章 文獻回顧…………………………………………………………………… 3
第一節 咖啡酸苯乙酯…………………………………………….…………... 3
2.1.1 咖啡酸苯乙酯簡介………………………………………...…. 3
2.1.2 咖啡酸苯乙酯與蜂膠之生物活性………………………….... 3
2.1.3 咖啡酸苯乙酯與蜂膠對免疫調節之影響………………….... 5
第二節 免疫系統………………………………………………………….…… 6
2.2.1 先天性與適應性免疫…………………………………...……. 6
2.2.2 輔助型T細胞分泌之細胞激素類型……………………….... 7
2.2.3 過敏反應的發生……………………………………………… 8
第三節 氣喘……………………………………………………………………. 8
2.3.1 氣喘簡介……………………………………………………… 8
2.3.2 氣喘發生的機制……………………………………………… 9
2.3.3 發炎相關之化學趨化因子………………………………….. 10
2.3.4 治療氣喘的方式…………………………………………….. 10
第四節 氣喘動物模式之建立………………………………………………... 11
第三章 研究動機與目的…………………………………………………………... 13
第四章 實驗設計…………………………………………………………………... 15
第五章 材料與方法………………………………………………………………... 16
第一節 探討CAPE對氣喘模式小鼠之活體外 (ex vivo) 試驗：
CAPE對氣喘模式小鼠淋巴細胞之影響…………………………… 16
5.1.1 試驗物質CAPE配製………………………………………. 16
5.1.2 氣喘模式動物的建立……………………………………….. 19
5.1.3 實驗動物犧牲……………………………………………….. 20
5.1.4 將CAPE與分離自氣喘模式動物之活體細胞進行
體外實驗……………………………………………………..21
5.1.5 血清中抗體與細胞培養上清液中細胞激素之測定……….. 23
第二節 CAPE在in vitro條件下調控Jurkat T細胞株分泌IFN-
之情形………………………………………………………………... 26
5.2.1 將Jurkat T細胞進行電穿孔 (Electroporation)………….. 26
5.2.2 Jurkat T細胞、T細胞之活化劑或CAPE共同培養
之條件………………………………………………………. 27
5.2.3 分析Jurkat T細胞中透過質體轉入的IFN-表現量……… 28
第三節 CAPE之活體試驗：探討餵食CAPE對氣喘模式動物免疫
調節作用之影響……………………………………………………... 29
5.3.1 實驗動物…………………………………………………….. 29
5.3.2 實驗動物犧牲……………………………………………….. 31
5.3.3 免疫功能相關之測定……………………………………….. 34
5.3.4 實驗試劑與配製…………………………………………….. 40
第四節 統計方法……………………………………………………………... 42
第六章 結果與討論………………………………………………………………... 43
第一節 探討CAPE對氣喘模式小鼠之活體外 (ex vivo) 試驗：
CAPE對氣喘模式小鼠淋巴細胞之影響…………………………....43
6.1.1 CAPE對氣喘模式小鼠腹腔細胞培養上清液中促發炎
　　　 細胞激素分泌量之影響…………………………………... 43
6.1.2 CAPE對氣喘模式小鼠脾臟細胞培養上清液中細胞激素
分泌量之影響…………………………………………..44
6.1.3 CAPE對氣喘小鼠脾臟細胞增生反應之影響………………45
第二節 CAPE在in vitro條件下調控Jurkat T細胞株分泌IFN-之
情形……………………………………………………………………47
第三節 CAPE之活體試驗：探討餵食CAPE對氣喘模式動物免疫
調節作用之影響…………………………………………………….. 48
6.3.1 餵食CAPE對氣喘模式小鼠生長體重之影響 48
6.3.2 餵食CAPE對氣喘模式小鼠血清中OVA特異性
　　　　　　　　IgE抗體生成量之影響……………………………………49
　　　　　　6.3.3 餵食CAPE對氣喘模式小鼠肺部沖洗液中免疫相關
　　　　　　　　　　分析之影響………………………………………………...49
　　　　　　6.3.4 餵食CAPE對氣喘模式小鼠腹腔細胞培養上清液中
　　　　　　　　　　促發炎細胞激素分泌量之影響……………………….......50
　　　　　　6.3.5 餵食CAPE對氣喘模式小鼠脾臟細胞免疫相關分析
　　　　　　　　　　之影響………………………………………………………51
第七章 總結…………………………………………………………………………54
圖………………………………………………………………………………………56
參考文獻………………………………………………………………………………92


圖 目 錄

圖一 OVA致敏之氣喘模式動物建立之流程……………………………… 56
圖二 CAPE對氣喘小鼠腹腔細胞培養上清液中IL-6分泌量之影響……. 57
圖三 CAPE對氣喘小鼠腹腔細胞培養上清液中TNF-分泌量之影響…. 58
圖四 CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液中
　　　　　　IFN-分泌量之影響…………………………………………………. 59
圖五 CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液中
　　　　　　IL-4分泌量之影響…………………………………………………...60
圖六 CAPE對氣喘小鼠脾臟細胞以不同有絲分裂劑刺激培養
　　　　　　增生反應之影響…………………………………………………….. 61
圖七 CAPE對氣喘小鼠脾臟細胞以OVA抗原刺激培養增生反
　　　　　　應之影響……………………………………………………………...63
圖八 不同濃度CAPE對Jurkat cells之IFN-基因表現的影響………… 64
圖九 CAPE之活體試驗流程圖……………………………………………. 66
圖十 CAPE對小鼠生長體重之影響………………………………………. 67
圖十一 餵食CAPE對於氣喘小鼠血清中OVA特異性IgE抗體生
　　　　　　成量之影響………………………………………………………….. 68
圖十二 餵食CAPE對於氣喘小鼠肺部沖洗液中發炎細胞巨集情形
　　　　　　之影響……………………………………………………………….. 69
圖十三 餵食CAPE對於氣喘小鼠肺部沖洗液中IL-5分泌量之影響……… 71
圖十四 餵食CAPE對於氣喘小鼠肺部沖洗液中Eotaxin分泌量之
影響…………………………………………………………………… 72
圖十五 餵食CAPE對於氣喘小鼠肺部沖洗液中MCP-1分泌量之
影響…………………………………………………………………… 73
圖十六 餵食CAPE對氣喘小鼠腹腔細胞以LPS刺激培養上清液
　　　　　中IL-6分泌量之影響…………………………………………………. 74
圖十七 餵食CAPE對氣喘小鼠腹腔細胞以LPS刺激培養上清液
　　　　　中TNF-分泌量之影響……………………………………………….. 75
圖十八 餵食CAPE對氣喘小鼠腹腔細胞以LPS刺激培養上清液
　　　　　中IL-1β分泌量之影響……………………………………………….. 76
圖十九 餵食CAPE對氣喘小鼠腹腔細胞以LPS刺激培養上清液
　　　　　中MCP-1分泌量之影響……………………………………………… 77
圖二十 餵食CAPE對氣喘小鼠脾臟細胞以不同有絲分裂劑刺激增
　　　　　生反應之影響………………………………………………………….. 78
圖二十一 餵食CAPE對氣喘小鼠脾臟細胞以不同濃度OVA抗原刺激
　　　　　增生反應之影響……………………………………………………….. 80
圖二十二 氣喘小鼠脾臟細胞以PMA與ionomycin刺激培養6小時後，
　　　　　會分泌IFN- (A)或IL-4 (B) 的CD3 T細胞百分比………………… 81
圖二十三 餵食CAPE對氣喘小鼠脾臟細胞(B)或CD3 T細胞(A)內
IFN-之表現情形……………………………………………………... 82
圖二十四 餵食CAPE對氣喘小鼠脾臟細胞(B)或CD3 T細胞(A)內
IL-4之表現情形……………………………………………………… 84
圖二十五 餵食CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液
　　　　　中IFN-分泌量之影響………………………………………………… 86
圖二十六 餵食CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液
　　　　　中IL-4分泌量之影響…………………………………………………. 87
圖二十七 餵食CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液
　　　　　中IL-5分泌量之影響…………………………………………………. 88
圖二十八 餵食CAPE對氣喘小鼠脾臟細胞以Con A刺激培養上清液
　　　　　中IL-10分泌量之影響………………………………………………... 89

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