中草藥(甘露飲)對口腔癌細胞離體與活體之免疫調節影響; The immunomodulating effects of the Chinese herbal medicine Gan-Luh-Yin (GLY) on oral cancer cells：in vitro and in vivo

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 生命科學院 > 生物科技學系暨碩士班 > 博碩士論文 > Item 310903500/514

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题名:	中草藥(甘露飲)對口腔癌細胞離體與活體之免疫調節影響;The immunomodulating effects of the Chinese herbal medicine Gan-Luh-Yin (GLY) on oral cancer cells：in vitro and in vivo
作者:	吳家珺;Chia-Chun Wu
贡献者:	中國醫藥大學：生物科技學系碩士班
关键词:	甘露飲;口腔癌;抗發炎反應;免疫調節;原位注射口腔癌模式;Gan-Luh-Yin (GLY);Oral cancer;anti- inflammation;immunomodulation;orthotopic animal model
日期:	2009-05-25
上传时间:	2009-08-11 10:58:36 (UTC+8)
摘要:	口腔癌是目前台灣常見的癌病變，在癌症病患的血液中分析顯示T細胞、B細胞與IL-2較正常人低，而IL-4，IL-6與TNF是比一般正常人高。甘露飲具有調整機體抗病能力，為臨床上治療口腔癌病人放射療法後中醫師所給予病人的常見輔助方劑，本研究主要是探討甘露飲在口腔癌離體與活體上免疫調節功能的影響。在離體實驗上，以MTS的方法檢測甘露飲對於B細胞與T細胞之增生作用，以CBA方法與FACScan分析細胞激素(IL-2、IL-4、IL-6、IL-10、TNF與IFN-γ)之分泌，也用即時聚合?○s鎖反應偵測 TNF-α的 mRNA 變化及西方墨點法檢測TNF-α與NF-κB、AKT/PKB、ERK 三大路徑相關性，並以免疫螢光染色法與EMSA觀察NF-κB與DNA結合的能力，最後以NF-κB 啟動子分析測定NF-κB 的活性；而在活體實驗上，以BALB/c 裸小鼠原位注射的方式建立誘發口腔癌模式，並觀察甘露飲對口腔癌裸鼠飲水量之影響；接著也觀察甘露飲對BALB/c小鼠免疫功能之影響，以MTS 方法檢測BALB/c小鼠脾臟細胞增生作用，以流式細胞儀分析巨噬細胞吞噬能力、自然殺手細胞毒殺作用、細胞表面抗原與細胞激素的檢測。結果顯示，甘露飲200、500、1000 μg/ml能促進BALB/c小鼠離體脾臟細胞經由Con A與LPS刺激之後T、B細胞的增生。由CBA分析結果顯示，甘露飲2000 μg/ml最能顯著抑制CAL-27之TNF-α分泌，且隨著作用時間增長，甘露飲2000 μg/ml能抑制TNF-α的mRNA表現，並調節核內NF-κB p65、p50與上游phospho-IKK (Ser176) 、phospho- IκB及p-AKT、PI3K與ERK、p-ERK這三條路徑蛋白質表現。然而，甘露飲也抑制NF-κB p65在細胞核的表現及NF-κB活性。在活體實驗結果顯示，甘露飲具有降低口腔癌BALB/c裸小鼠的飲水量，因此證實甘露飲具有減少口腔癌動物口乾舌燥之副作用；而在BALB/c小鼠體重、脾臟與肝臟並沒有影響。經餵食甘露飲後，具有提升脾臟細胞對Con A與LPS刺激增生之能力，並顯著的增強周邊血液中巨噬細胞吞噬能力，更提升T細胞的增生。此外，甘露飲也具有降低細胞激素TNF-α的效果。綜合以上結果，我們推論甘露飲能作為口腔癌細胞輔助治療的藥劑，經離體與活體的實驗結果推測可能是藉由降低細胞激素TNF-α之影響，以及在活體上具有調節免疫功能。 Oral cancer is one of the major cancers in Taiwan. It was reported that the number of T and B cells and the levels of IL-2 in oral cancer patients are lower than normal subjects. However, the level of IL-4, IL-6 and TNF-α in oral cancer patients are higher. Gan-Luh-Yin (GLY) is usually used for promoting cure of Chinese herbal medicine in clinical patients. However, the effects of GLY anti-inflammation and immunomodulation are not clear in oral cancer. The purpose of this study is to investigate the mechanisms of GLY via anti- inflammation and immunomodulation. We measured the proliferation of T and B cells by Celltiter96 kit. And the stimulation of cytokines, the cell populations in peripheral blood mononuclear cell (PBMC), the activity of phagocytosis in macrophage and cytotoxic ability in nature killer (NK) cell were detected by Flow cytometer. The cytokines regulated proteins were determined by Western blotting and immunofluorescence staining. And the expression of nuclear factor kappa B (NF-κB) mRNA was measured by RT-PCR. We also used promoter assay and EMSA for determent the DNA binding ability in HNSCC cell line. In vitro, the results indicate that GLY in 200, 500 and 1000 μg/mL can promoted the proliferation of T and B cells under Con A and LPS stimulation, respectively. The result from CBA and FACS can examination indicated that: GLY can inhibit the secretion of TNF from CAL27, SCC-4, HSC-3 and TW206 cell. Beside, GLY suppressed TNF mRNA level by Real-time PCR. GLY also suppressed expression of proteins of the Akt phosphorylation, NF-κB p65 and ERK1/2. The translocation and DNA binding ability of NF-κB were decreased in CAL27 cell. In vivo, we treated BALB/c Nude mice and BALB/c mice with different concentrations of GLY. Our results showed that GLY were not effect on the body, liver and spleen weights of BALB/c mice. GLY could decrease the cytokines levels of TNF-α, and increase the levels of T cell and macrophage population in PBMC. And it also increased macrophage phagocytosic activity. In addition, GLY also increased the spleen cells proliferation when stimulated by Con A. GLY also reduced the volume of drinking water in BALB/c Nude mice orthotopic animal model. Taken together, we suggested GLY can promote anti-inflammatory in oral cancer may through effect on the levels of TNF-α. And the mechanisms of immune- respones were caused by increase the population of T cells and macrophage phagocytosic activity, then decrease the level of TNF-α in vivo.
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