以人類周邊血液分離培養樹狀細胞探討檳榔成份之免疫調節作用

本實驗使用人類周邊血液單核球(peripheral blood monocytes)分化為未成熟樹狀細胞（immature dendritic cell；iDC）的實驗模式，探討檳榔萃取物（areca nut extract；ANE）和檳榔鹼（arecoline；AR）對DC分化（differentiation）及反應性之影響。在DC分化方面，PBMC加入ANE 10 - 20 mg/mL或AR 10 - 100 mM培養7天後，分化成為iDC的數量顯著較未處理藥物之對照組少，且ANE使已分化之iDC的存活率明顯降低。以流式細胞儀分析PBMC培養7天後之細胞表面抗原，圈選體積較大、顆粒性較高之細胞分群進行分析，結果顯示ANE 10 mg/mL及AR 100 mM處理使CD1a的表現減少，ANE 20 mg/mL處理使CD1a 、CD40及CD86的表現減少；相同實驗以流式細胞儀分析未經圈選的PBMC培養7天後之全體細胞，ANE 20 mg/mL處理使CD40的表現減少，AR 100 mM處理使HLA-DR及CD1a的表現減少，但AR 10、100 mM處理則使CD86的表現上升。在iDC的細胞形態方面， ANE 25 mg/mL及AR 500 mM以上濃度在24小時內使細胞呈現圓形化，但以MTT試驗法檢測iDC的細胞代謝活性，則發現在24小時作用下LPS 10 ng/mL、ANE 10 - 200 mg/mL及AR 10 mM對細胞代謝活性均無影響，只有AR 50 mM以上濃度使細胞代謝活性下降。在iDC的反應性方面，iDC以ANE 10、20 mg/mL及AR 10、100 mM預先處理30分鐘，再加入LPS 10 ng/mL刺激24小時，以流式細胞儀檢測體積較大、顆粒性較高之細胞分群，結果顯示藥物處理組與LPS刺激組的表面抗原表現無顯著差異。iDC的反應性以反轉錄聚合酶鏈連鎖反應（RT-PCR）檢測IL-12B（p40）mRNA的表現，結果顯示LPS明顯刺激iDC 表現IL-12B，而此表現會受到ANE 10、20 mg/mL及AR 100 mM的抑制。綜合上述，本研究指出ANE及AR對於人類DC之分化及其反應性具有明顯影響，除了使分化的iDC細胞數量減少，表面抗原表現傾向分化不完全以外，ANE與AR對於iDC的反應性亦有顯著的抑制作用。

關鍵字

檳榔萃取物；樹狀細胞；成熟；檳榔鹼；脂多醣；免疫調控；分化；介白素-12

並列摘要

The effect of areca nut extracts (ANE) and arecoline (AR) on the differentiation and activation of dendritic cells (DC) was investigated in the present studies. Monocytes isolated from human peripheral blood were cultured in RPMI medium supplemented with GM-CSF and IL-4 to generate DC. Several lines of evidence suggested that the differentiation of DC from monocytes was suppressed by the presence of ANE or AR. First, the cell number of immature DC obtained following 7-day culture of monocytes was decreased in the presence of ANE (10 - 20 mg/mL) or AR (10 - 100 mM). Second, the viability of DC was decreased by ANE treatment. Lastly, the expression of surface markers of DC was influenced by ANE or AR treatment as determined by flow cytometry. The expression of CD40 on total harvested cells was decreased by ANE (20 mg/mL), and the expression of HLA-DR and CD1a was attenuated by AR (100 mM). Similar results were also observed in selected cell populations with larger size. The morphological studies revealed that a higher percentage of iDC remained in round shape in the presence of ANE (25 mg/mL) or AR (500 mM), as compared to the control group. However, the metabolic activity of iDC was not significantly affected by ANE treatment as measure by a MTT assay. In contrast, the metabolic activity of iDC was suppressed by treatments with AR (50 - 1000 mM). Furthermore, the effect of ANE and AR on the reactivity of iDC was studied. Both ANE and AR did not affect the expression of cell surface markers on iDC stimulated with LPS. However, the induction of IL-12B mRNA expression by LPS in iDC was markedly suppressed by pretreatment of cells with ANE (20 mg/mL) or AR (10 - 100 mM). Taken together, the present studies demonstrated that the differentiation of iDC from monocytes and the activation of iDC were significantly suppressed by the presence of ANE and AR.

並列關鍵字

areca nut extract ； interleukin-12 ； dendritic cell ； differentiation ； lipopolysaccride ； maturation ； arecoline ； immunomodulation

參考文獻

60. Hsu, H.J., Chang, K.L., Yang, Y.H. & Shieh, T.Y. The effects of arecoline on the release of cytokines using cultured peripheral blood mononuclear cells from patients with oral mucous diseases. Kaohsiung J Med Sci. 17, 175-82. (2001).

81. 蕭淑貞。檳榔子與大葉合歡樹皮單寧以及其相關化合物之研究。台北醫學大學碩士論文。1993。

61. Robinson PR, S.A., Knigh SC. Dendritic cell protocols. Human press otowa, New Jersey.( 2001).

78. Robinson PR, Stagg AJ, Knigh SC. Dendritic cell protocols. Human press, Totowa, New Jersey. 2001.

1. Banchereau, J. & Steinman, R.M. Dendritic cells and the control of immunity. Nature. 392, 245-52. (1998).

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以人類周邊血液分離培養樹狀細胞探討檳榔成份之免疫調節作用

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