詹東榮臺灣大學：獸醫學研究所王家琪Wang, Chia-ChiChia-ChiWang2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/59910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.中文摘要 I Abstract II 簡寫對照表 IV 目錄 V 圖次 VII 表次 VIII 第一章、前言與文獻探討 1 第一節、T淋巴球的功能及其凋亡機制 1 一、T淋巴球及其分泌之細胞激素在免疫系統的功能 1 二、免疫系統對抗腫瘤形成的機制 2 三、T淋巴球凋亡之機制 3 第二節、檳榔成份的致癌性與免疫機能惡化相關性之探討 4 一、檳榔子與檳榔嚼塊的主成分 4 二、檳榔致癌機轉的探討 5 三、嚼食檳榔對免疫系統的影響 7 第二章、實驗材料與方法 10 第一節、藥品資料 10 第二節、實驗方法 12 一、實驗動物與細胞培養模式 12 二、酵素連結免疫吸附試驗(enzyme-linked immunosorbent assay ; ELISA) 12 三、細胞增生與MTT代謝活性的偵測 12 四、RT-PCR 13 五、Multi-probe RNase protection assay (RPA) 13 六、DNA fragmentation assay 14 七、Annexin V/propidium iodide染色分析細胞凋亡 14 八、測定粒腺體膜電位 (mitochondrial membrane protential；△Ψm) 15 九、脾臟細胞CD4+/CD8+ /B220+表面抗原 (surface marker)與annexin V分析 15 十、抗氧化劑對ANE作用之影響 16 十一、數據統計分析 16 第三章、實驗結果 17 第一節、檳榔成分對於T細胞存活、細胞代謝活性、活化及分泌細胞激素的影響 17 一、檳榔子水萃取物(ANE)對於小鼠脾臟細胞及EL4細胞存活率及代謝活性的影響 17 二、ANE對於T細胞分泌細胞激素的影響 17 三、比較ANE、檳榔生物鹼(arecoline及arecaidine)、多酚類檳榔萃取物 (polyphenolic ANE)及黃樟素(safrole)對於脾臟細胞代謝活性及分泌細胞激素的影響 18 第二節、ANE抑制T細胞活化、細胞代謝活性及細胞激素分泌機制的探討 28 一、以RT-PCR測定ANE對於細胞激素mRNA表現的影響 28 二、Annexin V和propidium iodide染色分析ANE誘發T細胞凋亡的發生 28 三、Annexin V和surface marker染色分析ANE誘導不同族群脾臟細胞凋亡的發生 34 四、分析ANE處理後細胞DNA ladder的發生 39 五、以RPA測定ANE處理後細胞表現caspase mRNA的變化 40 六、ANE處理對脾臟細胞粒腺體膜電位的影響 40 七、抗氧化劑對於ANE抑制細胞活化和存活率的保護效果 40 八、抗氧化劑對於ANE抑制分泌細胞激素的保護效果 40 第四章、討論 50 參考文獻 56 圖次 Figure 1. The effect of ANE, arecoline and arecaidine on splenocyte metabolic activity. 19 Figure 2. The effect of ANE and arecoline on the viability and metabolic activity of EL4 cells. 20 Figure 3. The effect of ANE, Arecoline and Arecaidine on splenocyte viability. 21 Figure 4. The effect of ANE on the viability and growth of EL4 cells. 22 Figure 5. The effect of ANE on the production of IL-2, IL-4 and IFN-γ induced by PMA plus ionomycin in splenocytes. 23 Figure 6. The effects of ANE on the production of IL-2 and IL-4 induced by PMA plus ionomycin in EL4 cells. 24 Figure 7. The effect of ANE, Polyphenolic ANE, Arecoline, Arecaidine and Safrole on the production of IL-2 induced by PMA plus ionomycin in splenocytes. 25 Figure 8. The effect of ANE, Polyphenolic ANE, Arecoline, Arecaidine and Safrole on the production of IFN-γinduced by PMA plus ionomycin in splenocytes. 26 Figure 9. The effect of ANE, Polyphenolic ANE, Arecoline, Arecaidine and Safrole on the production of IL-4 induced by PMA plus ionomycin in splenocytes. 27 Figure 10. The effect of ANE on IL-2, IL-4 and IFN-γ mRNA expression induced by PMA plus ionomycin in splenocytes. 29 Figure 11. The effect of ANE on IL-2 and IL-4 mRNA expression induced by PMA plus ionomycin in EL4 cells. 30 Figure 12. Concentration-dependent induction of apoptosis and necrosis by ANE in splenocyte. 31 Figure 13. Concentration-dependent induction of apoptosis and necrosis by ANE in EL4 cells. 32 Figure 14. Time-dependent induction of apoptosis and necrosis by ANE in EL4 cells. 33 Figure 15. Time-dependent induction of apoptosis of splenic CD4+ cells by ANE. 35 Figure 16. Time-dependent induction of apoptosis of splenic CD8+ cells by ANE. 36 Figure 17. Time-dependent induction of apoptosis of splenic B220+ cells by ANE. 37 Figure 18. Time-dependent induction of apoptosis of splenic (A) CD4+, (B) CD8+ and (C) B220+ by ANE. 38 Figure 19. Induction of chromosomal DNA fragmentation in EL4 cells and splenocytes by ANE. 39 Figure 20. The effect of ANE on the steady state mRNA expression of caspases by EL4 cells. 42 Figure 21. The effect of ANE on the steady state mRNA expression of caspases by EL4 cells. 43 Figure 22. Time-dependent induction by ANE of the depolarization of mitochondrial membrane potential in splenocytes. 44 Figure 23. Attenuation of ANE-mediated inhibition of splenocyte metabolic activity by N-acetyl-cysteine (NAC). 45 Figure 24. Attenuation of ANE-mediated inhibition of splenocyte metabolic activity by glutathione (GSH). 46 Figure 25. Attenuation by NAC of ANE-mediated inhibition of IFN-γ production by splenocyte. 47 Figure 26. Attenuation by NAC of ANE-mediated inhibition of IL-2 production by splenocyte. 49 表次 Table 1. Attenuation by NAC, glutathione (GSH) and superoxide dismutase (SOD) of ANE-mediated inhibition of IFN-γ production by splenocytes. 48885956 bytesapplication/pdfen-UST淋巴球細胞激素細胞凋亡檳榔氧化性傷害T lymphocytecytokineInterferonapoptosisareca nutreactive oxygen speciesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59910/1/ntu-94-R92629014-1.pdf