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標題: | 檳榔子水萃取物抑制T細胞活化和引起細胞凋亡之毒性作用 Inhibition of T-cell activation and induction of apoptosis by areca nut extract |
作者: | Chia-Chi Wang 王家琪 |
指導教授: | 詹東榮(Ton-Rong Jan) |
關鍵字: | T淋巴球,細胞激素,細胞凋亡,檳榔,氧化性傷害, T lymphocyte,cytokine,Interferon,apoptosis,areca nut,reactive oxygen species, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 嚼食檳榔的習慣普遍流行在東南亞及台灣地區,流行病學研究顯示嚼食檳榔會提高罹患口腔癌的機率,在口腔癌病人中可以觀察到T細胞媒介免疫機能惡化的現象。本實驗主要研究檳榔子水萃取物 (areca nut extract; ANE) 對於離體T細胞功能的影響,並探討其毒性作用的可能機制。ANE (20-60 mg/mL) 抑制小鼠脾臟細胞及EL4細胞分泌IL-2 (T細胞活化的指標),TH1細胞激素IFN-g受到ANE的抑制作用比IL-2更為敏感,於ANE 10 mg/mL以上的濃度即呈現明顯的抑制作用。相反的,ANE對於 TH2細胞激素IL-4的影響則較不明顯。ANE (40-60 mg/mL) 對於脾臟細胞和EL4細胞的代謝活性及增生也呈現濃度相關性的抑制作用。然而給予高達100 μM的檳榔生物鹼 (arecoline 和 arecaidine) 處理,對於細胞存活率和細胞激素的表現並沒有作用,顯示ANE的作用並非由檳榔生物鹼所造成。進一步以RT-PCR分析ANE對於T細胞表現IL-2和 IFN-g mRNA的影響,發現ANE對mRNA的作用不如抑制蛋白質分泌的程度顯著。以annexin V和propidium iodide染色,使用流式細胞儀測定細胞的凋亡反應,發現ANE (20-40 mg/mL) 處理細胞三小時即可觀察到細胞出現凋亡的比率增加,以rhodamine123 染色分析,結果顯示ANE 40 mg/mL處理下,細胞粒腺體膜電位也發生去極化的改變。進一步分析ANE處理細胞的DNA,隨著ANE濃度增加,DNA laddering現象也變得更為明顯。以RNase protection assay 測定EL4細胞 caspase mRNA的表現,發現ANE (20 mg/mL) 處理細胞4小時caspase-2 mRNA 的表現上升。脾臟細胞給予抗氧化劑 N-acetyl-cysteine (NAC, 1-4 mM)、glutathione (GSH, 1-4 mM) 及superoxide dismutase (SOD, 1-400 units) 前處理,對於ANE抑制IL-2和代謝活性的作用有部份的回復效果,對於ANE抑制IFN-g的作用有更顯著的回復效果。
綜合上述,ANE具抑制T細胞活化、增生和誘發凋亡反應的作用,對於TH1細胞激素IFN-g的抑制程度比TH2細胞激素IL-4明顯,上述ANE抑制T細胞的作用可以被抗氧化劑部分回復,顯示ANE的作用可能是經由增加細胞氧化性傷害及誘導T細胞的凋亡反應所造成,而檳榔子中的生物鹼並不是造成ANE抑制T細胞功能的主要成份。 Betel quid chewing is an etiologic factor for oral squamous cell carcinoma (OSCC). Experimental evidence suggests that impaired immune reactivity was associated with the occurrence of OSCC. The objective of the present studies was to investigate the effect of areca nut extract (ANE) on T-cell function and cytokine expression. Pretreatment of splenocytes with ANE suppressed the production of IL-2 and the T-helper 1 (TH1) cytokine IFN-γ induced by PMA/Io. ANE suppression of IL-2 secretion was also demonstrated in EL4 cells. In contrast, the steady state mRNA expression of IL-2 and IFN-γ in splenocytes was not significantly inhibited by ANE. Moreover, ANE treatment produced a marked inhibition on the proliferation of splenocytes and EL4 cells as measured by a MTT assay. A 24-hr exposure of EL4 cells to ANE resulted in a marked reduction in cell viability and growth rate. Treatment of splenocytes and EL4 cells with ANE also increased the percentage of apoptotic cells, as evidenced by annexin V/ propidium iodide flow cytometry and DNA laddering. For mechanistic studies, the expression of various caspase mRNA in EL4 cells was simultaneously determined by a RNase protection assay. The steady state mRNA expression of caspase-2 was enhanced at 4 hr after ANE treatment. Depolarization of mitochondrial membrane potential in T cells was demonstrated by a decrease in rhodamine123 fluorescence after ANE treatment. The role of reactive oxygen species (ROS) in ANE-mediated effects on cytokine production was further investigated. Pretreatment of splenocytes with anti-oxidants, including N-acetyl-cysteine, superoxide dismutase and glutathione, partially attenuated the effect of ANE on T cell metabolic activity and IL-2 production, whereas ANE-induced suppression of IFN-γ production was almost completely reversed by pretreatment with anti-oxidants. Taken together, results from the present studies demonstrated that ANE markedly suppressed the activity and cytokine production by T cells, which was associated with the induction of apoptosis in T cells. In addition, the mechanistic studies suggested that the inhibitory effect of ANE on T cell activity and cytokine production was mediated, at least in part, via the induction of ROS in T cells by ANE. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24308 |
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顯示於系所單位: | 獸醫學系 |
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