詹東榮臺灣大學:獸醫學研究所郭馥華Kuo, Fu-HuaFu-HuaKuo2010-05-042018-07-092010-05-042018-07-092008U0001-2407200812514700http://ntur.lib.ntu.edu.tw//handle/246246/178912水飛蓟賓 (silibinin) 和水飛蓟 (silymarin) 用於治療肝臟疾病已經有相當久的歷史 (Gazak t al, 2007)，近年來，更廣泛作為保健食品或是醫療用途的藥品製劑，包含臺灣 。截至目前為止，學文獻的研究報告證實水飛蓟賓和水飛蓟具有許多有潛力的生物活性作用，其中包含對免疫的節作用，例如影響 T 細胞的功能及其細胞激素基因的表現。然而，有關水飛蓟賓和水飛蓟對h1/Th2免疫平衡的影響，現有的文獻報告之結果並不ㄧ致，仍有待進一步釐清。因此，本論文主旨在研究水飛蓟賓對Th1/Th2 免疫反應的作用，採用包含離體細胞培養和活體動物模式，探水飛蓟賓對 T 細胞參與的抗體生成、呼吸道過敏性免疫反應和細胞激素基因表現的影響。實結果顯示，在卵白蛋白(ovalbumin；OVA)免疫的 BALB/c 小鼠，於免疫前連續三天口服投予飛蓟賓，具有促進脾臟細胞表現 IFN-γ和血清中OVA 專一性IgG2a 抗體生成的作用；而且，脾細胞表現IL-4 和血清中OVA 專一性抗體IgE及total IgE 則受到水飛蓟賓的抑制。這些證據顯口服水飛蓟賓會使OVA 免疫小鼠的 T 細胞免疫反應，傾向Th1 的方向。本論文進一步採用以VA氣霧刺激OVA 免疫小鼠之呼吸道過敏的動物模式，結果發現在免疫前以及氣霧刺激前口服予水飛蓟賓，對呼吸道所引起的過敏性發炎反應沒有影響，但有促進肺臟組織中IFN-γ mRNA現的效果。在離體細胞培養的實驗模式，以水飛蓟賓處理由 OVA 免疫小鼠分離的脾臟細胞，現對 IFN-γ和 IL-4 皆有抑制作用，這些結果顯示在離體細胞直接暴露於水飛蓟賓的實驗條件，水飛蓟賓對於脾臟細胞表現細胞激素基因的作用和活體給藥的結果不同。 本論文採用BALB/c，嘗試了二種動物模式活體投予水飛蓟賓，和一種離體細胞模式處理水飛蓟賓，所得實驗結果然欠缺一致的結論，分析水飛蓟賓在現有文獻報告中，對 Th1/Th2 的影響亦有許多分歧的實驗據，顯示水飛蓟賓對 T 細胞免疫反應作用之複雜，仍有待進一步加以釐清。值得一提的是，本論文的離體試驗模式下，水飛蓟賓在較低的濃度(5-10 μM)下，即對Th1 (IFN-γ)和Th2 (IL-4)胞激素有明顯的影響，合併活體給藥的實驗結果，明顯指出 T 細胞是水飛蓟賓的作用標的之，其影響細胞激素基因表現的機轉值得未來加以深入探討。Silibinin and silymarin have been used as herbal medicines to treat liver disease for a long time. ecent years, silibinin and silymarin has been commonly used as dietary supplements, as well as edicinal products in many countries, including Taiwan. To date, scientific evidence indicates that ilibinin and silymarin possess potential biological activities, including immune modulation (i.e. effects n T cell reactivity and cytokine expression). Although the immunomodulatory activity of silibinin on cell functionality has been documented, contrasting effects on the balance of T helper (Th)1/Th2 ell-mediated immunity were reported. Hence, the objective of the present study was to investigate the ffect of silibinin on Th1/Th2 immune balance. Both animal models and cell culture experiments were mployed to examine the influence of silibinin on T-dependent antibody production, T cell-mediated llergic airway responses, and cytokine expression. The results demonstrated that daily oral dministration of silibinin for 3 days prior to ovalbumin (OVA) sensitization markedly enhanced the roduction of IFN-γ by splenocytes and the serum level of OVA-specific IgG2a in OVA-sensitized ALB/c mice. In contrast, the production of IL-4 and OVA-specific IgE and total IgE was attenuated. hese finding indicates that silibinin administration polarizes the Th1/Th2 balance toward the Th1 irection. In the murine model of allergic airway responses induced by the challenge of OVA aerosol to VA-sensitized mice, no significant effect on the airway allergic immune response by silibinin dministration was observed; however, the steady state mRNA expression of IFN-γ in the lung tissues f OVA-sensitized and challenged mice was markedly enhanced by silibinin administration. urthermore, the direct effect of silibinin on T cell-derived cytokine expression was also studied in itro. Splenocytes obtained from OVA-sensitized mice were exposed to silibinin in culture and timulated with OVA to induce cytokine production. The results showed that silibinin exposure ignificantly attenuated the production of both IFN-γ and IL-4 by OVA-stimulated splenocytes. These esults demonstrated that the effects of silibinin on T cell cytokine expression between the employed in ivo and in vitro experimental settings are inconsistent. The present study utilized 3 experimental ystems, including animal models and cell culture experiments, to examine the immunomodulatory ffect of silibinin on T cells; however, the results on T cell cytokine expression are contrasting. The nconsistency of silibinin-mediated effects on the Th1/Th2 immune balance has been reported in the iterature, suggesting a very complicated profile of silibinin influence on the immune system. It is oticed that, under the employed condition of cell culture studies, the effective concentration range of IVilibinin (5-10 μM) to affect Th1 (IFN-γ) and Th2 (IL-4) cytokine expression is lower than the oncentrations used by many reports in the literature. Together with the demonstrated effects of ilibinin on Th1/Th2 cytokine expression in murine models, it is apparent that T cells are a sensitive arget for silibinin. Further studies to elucidate the mechanism of silibinin-mediated effects on cytokine xpression are warranted.致謝..............................................I 文摘要......................................... II bstract..........................................III 一章、前言與文獻探討............................................. 1 一節、水飛蓟賓 (silibinin) 的背景介紹及其應用價值......................................... 1 、水飛蓟賓的來源.............................................. 1 、水飛蓟賓的生物活性作用.............................................. 3 二節、水飛蓟賓對免疫系統的影響.............................................. 6 、抗原專一性的後天免疫 (adaptive immunity)..................................... 6 、水飛蓟賓的免疫調節作用.................................................. 7 三節、論文主旨............................................... 8 二章、實驗材料與方法................................................. 9 一節、藥品資料............................................................................................................................... 9 二節、實驗方法..............................................................................................................................11 、實驗動物來源..........................................................................................................................11 、動物處理..................................................................................................................................11 、離體實驗的脾臟細胞製備..................................................................................................... 15 、離體實驗之以酵素連結免疫吸附試驗(enzyme-linked immunosorbent assay; ELISA)測定經白刺激所產生的細胞激素(IL-2、IL-4 和IFN-γ) .................................................................... 15 、離體實驗之以MTT 測定水飛蓟賓對細胞細胞增生的影響.............................................. 16 、動物實驗之血清分離............................................................................................................. 16 、以酵素連結免疫吸附試驗測定OVA 專一性抗體.............................................................. 16 、以酵素連結免疫吸附試驗測定OVA IgE 及total IgE......................................................... 17 、活體實驗之脾臟細胞製備及以酵素連結免疫吸附試驗測定經卵白刺激所產生的細胞動素IL-2、IL-4 和IFN-γ) .................................................................................................................... 17 、活體實驗之以MTT 偵測細胞增生變化.............................................................................. 17 ㄧ、活體實驗之脾臟細胞CD4+CD8+/B220+面抗原 (surface marker)分析.................... 18 二、解剖以及肺組織製備......................................................................................................... 18 三、反轉錄聚合酶鏈式反應..................................................................................................... 18 四、數據統計分析..................................................................................................................... 19 三章、實驗結果................................................................................................................................. 20 一節、離體細胞培養模式下水飛蓟賓對T細胞代謝活性及細胞激素表現的作用................ 20 、水飛蓟賓對於小鼠脾臟細胞代謝活性的影響..................................................................... 20 、水飛蓟賓對於T細胞分泌細胞激素的影響........................................................................ 20 二節、活體投予水飛蓟賓對T細胞表現細胞激素和抗原專一性抗體生成之影響................ 23 、水飛蓟賓對於BALB/c 小鼠的毒性影響............................................................................. 23 、水飛蓟賓對於OVA 專一性抗體(IgG1、IgG2a、IgM 和IgE)和total IgE 的影響............. 23 、水飛蓟賓對脾臟細胞增生反應之影響................................................................................. 24 、以surface marker 染色分析水飛蓟賓對脾臟細胞族群的影響........................................... 24 、以ELISA測定水飛蓟賓對於T細胞分泌細胞激素........................................................... 24 三節、水飛蓟賓對小鼠呼吸道過敏性免疫反應的影響............................................................. 31 、水飛蓟賓對OVA 誘發呼吸道過敏反應的BALB/c 小鼠的毒性影響............................... 31 、水飛蓟賓對於total IgE的影響............................................................................................. 31 、水飛蓟賓對於脾臟細胞的增生反應之影響......................................................................... 31 VI、以ELISA測定水飛蓟賓對於T細胞表現細胞激素之影響............................................... 31 、以RT-PCR測定水飛蓟賓對於肺臟細胞激素 mRNA表現的影響.................................... 32 、以H&E染色觀察水飛蓟賓對於浸潤在肺臟的炎症細胞的影響...................................... 32 、以Alcian Blue (pH 2.5)-periodic acid-Schiff sequence (AB-PAS)染色觀察水飛蓟賓對於肺黏液分泌的影響......................................................................................................................... 32 四章、討論......................................................................................................................................... 40 一節、水飛蓟賓在卵白蛋白 (OVA) 免疫的BALB/c 小鼠對T細胞表現細胞激素和抗原專一抗體生成之影響............................................................................................................................. 40 二節、水飛蓟賓對BALB/c 小鼠以卵白蛋白 (OVA) 誘發呼吸道過敏模式之全身性和局部性影響................................................................................................................................................. 41 三節、水飛蓟賓對卵白蛋白免疫 (OVA-sensitized)T細胞之影響........................................... 43 五章 參考文獻................................................................................................................................... 45application/pdf750637 bytesapplication/pdfen-US水飛蓟賓T細胞卵白蛋白干擾素-γ白介素-4免疫球蛋白G2a免疫球蛋白EsilibininT cellovalbumininterferon-gammainterleukin-4IgG2aIgEthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178912/1/ntu-97-R95629026-1.pdf