2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38081Studies of human subjects have shown that chronic ethanol ingestion is associated with abnormalities of humoral and cellular immunity. These abnormalities may contribute to the increased incidence of infectious diseases and even cancer in alcoholics. Chronic ethanol administration also damages a broad spectrum of immune functions in experimental animals. Binge drinking of ethanol is probably more common than chronic heavy drinking, and this has become a serious problem among young persons. While chronic effects of ethanol on the immune system have been studied extensively, less information is available regarding immunosuppressive effects of acute ethanol exposure. Consumption of sufficient ethanol to produce blood levels greater than 0.2% causes a neuroendocrine stress response in humans and in rodent models. Similar stress responses to other stimuli are associated with decreased lymphocyte activity. Our previous studies have shown that a single dose of ethanol produces a substantial stress response in B6C3F1 mice and natural killer cell activity is suppressed by glucocorticoids and catecholamines produced as a consequence of this stress response. Macrophages play an important role in host resistance to microbes and tumor cells. The sensitivity of various lymphocytes to the same stress response may be different. It also has been reported that ethanol decreases human monocyte function via induction of transforming growth factor-???nand prostaglandin E2. Macrophages can be stimulated by a variety of reagents. The activated macrophages could kill more microbes and tumor cells. In this study we plan to use thioglycollate as stimulator for macrophages. Therefore, the present project will evaluate the effects of ethanol on mouse resident and inflammatory macrophages after a single dose of ethanol by gavage. Also, this project will focus the role of neuroendocrine mediators and/or the other mediators induced by ethanol involved in suppressing macrophage function and in inducing macrophage apoptosis. With microarray analysis, the gene expression profiling of acute ethanol responses also will be evaluated. This project may take five years to finish the following specific aims: 1) Evaluate the effects of ethanol on macrophage function. 2) Determine the role of ethanol-induced mediators in suppressing macrophage function. 3) Evaluate the signaling mechanisms involved in ethanol-induced macrophage apoptosis. 4) Determine the role of ethanol-induced mediators in inducing macrophage apoptosis. 5) Evaluate the genes induced by ethanol involved in altering macrophage survival and function with microarray approach. In the present study, the immune functions of macrophages including phagocytosis, hydrogen peroxide release, nitric oxide release, tumor necrosis factor-?? secretion, interleukin-12 secretion, MHC II molecules expression, and anti-P815 tumor cells cytotoxicity will be assayed. The apoptosis assay of macrophages will be analyzed by 4,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. Also, we will use inhibitors of glucocorticoids, catecholamines, opioids, cyclooxygenase-2 or transforming growth factor-?? antibody to treat mice to identify which mediator induced by ethanol may play a role in suppressing macrophage function and in inducing macrophage apoptosis. Finally, we will use commercial available gene chip to analyze the possible genes induced by ethanol involved in alterations of macrophage activity. The results of this project may have implications for human health, because even moderate ethanol consumption increases the incidence of infectious diseases and cancers. The data of this project could open an avenue of research on pharmacological interventions to block those aspects of stress responses根據研究顯示慢性酒精(ethanol)中毒者和有酒癮者罹患傳染病的機會比一般非酗酒者還要高，原因在於這些酗酒者的免疫功能已被抑制，故較不能抵抗外來的疾病。另有證據指出在一夕之間飲酒狂歡的人數並不少於慢性酒精中毒者，然而有關免疫功能在這些急性酒精中毒者的情形所知仍有限，故本人曾利用動物模式探討單一劑量的酒精對自然殺手細胞的影響。當飲酒到達一定量其血中酒精濃度超過0.2% 便會造成一種腦內性的緊迫反應 (stress response)，在此緊迫反應之下，一些神經內分泌物質 (neuroendocrine mediators) 分泌會增高導致免疫功能的下降。本人以前的研究顯示餵與單一劑量的酒精在老鼠體內可以抑制自然殺手細胞 (natural killer cells) 的功能，酒精所誘導的糖皮質素 (glucocorticoids) 和兒茶酚胺(catecholamines) 是抑制自然殺手細胞功能的主因。巨噬細胞 (macrophages) 在疾病的防禦上扮演一重要的角色，其對緊迫反應的敏感性不得而知。另有研究指出酒精所誘導的前列線素E2 (PGE2) 和細胞轉形成長因子？？？n(TGF-？？) 可抑制人類單核球 (monocytes) 的功能。故本計劃將探討急性酒精暴露對一般的和活化的老鼠巨噬細胞的影響。巨噬細胞可被多種刺激物所活化，這些被活化後的巨噬細胞比一般的巨噬細胞更能有效的殺死微生物或腫瘤細胞，因此被活化後的巨噬細胞比一般的巨噬細胞扮演更重要的免疫防禦功能。在本計劃下巨噬細胞將被一種物質叫thioglycollate所活化，這些被thioglycollate所活化後的巨噬細胞功能和被微生物所活化後的情形十分類似。本計劃將探討急性酒精暴露如何影響巨噬細胞，藉由誘發何種神經內分泌物質或細胞激素 (cytokines) 進而導致巨噬細胞免疫功能的下降。另外本計劃將探討是否急性酒精暴露可促進老鼠巨噬細胞發生細胞凋亡 (apoptosis) 及其可能涉及的分子機理。本計劃擬以五年的時間完成以下的研究目標： 一、 探討急性酒精暴露對老鼠巨噬細胞免疫功能的影響 二、 評估何者神經內分泌物質或細胞激素在急性酒精誘導下可抑制老鼠巨噬細胞的免疫功能 三、 探討急性酒精暴露對老鼠巨噬細胞發生細胞凋亡所涉及的分子機理 四、 評估何者神經內分泌物質或細胞激素在急性酒精誘導下可促進老鼠巨噬細胞發生細胞凋亡 五、 利用微矩陣晶片探討急性酒精誘導之基因涉及抑制老鼠巨噬細胞的免疫功能及促進老鼠巨噬細胞發生細胞凋亡 本實驗之巨噬細胞免疫功能分析包括吞噬能力 (phagocytosis)、釋放一氧化氮 (nitric oxide) 能力、釋放過氧化氫 (hydrogen peroxide) 能力、釋放腫瘤壞死因子 (tumor necrosis factor-？？) 能力、釋放介白質12 (Interleukin-12)、組織相容蛋白 (MHC II molecules) 表現及毒殺腫瘤細胞能力 (tumor lytic function)。在內分泌物質及細胞激素的實驗上，我們將以糖皮質素 (glucocorticoids)、兒茶酚胺 (catecholamines)、鴨片 (opioids)、前列線素E2的阻斷劑或細胞轉形成長因子 (transforming growth factor-？？) 抗體處理老鼠，以確認何種內分泌物質或細胞激素在巨噬細胞數目及其免疫功能方面扮演重要的角色。在細胞凋亡的實驗上，我們將以DAPI染色和流式細胞分析儀分析腹腔巨噬細胞發生細胞凋亡的百分比。另外我們將以西方墨點法 (Western blotting) 和酵素活性測試 (ELISA) 分析致凋亡蛋白 (proapoptotic proteins) 及caspases 在巨噬細胞發生細胞凋亡上所扮演的角色。最後我們將使用商業化之DNA晶片探討急性酒精誘導之基因涉及抑制老鼠巨噬細胞的免疫功能及促進老鼠巨噬細胞發生細胞凋亡。本計劃對急性酒精中毒者和酒癮者的健康情形將有重要的幫助，因為這些病患的疾病罹患率及致癌率均比一般人高，這或許和巨噬細胞功能受到抑制有關。本計劃如以人體作為急性酒精暴露的實驗對象，其研究結果將可直接應用於酒精中毒者和酒癮者之各種治療，包括免疫治療。然而人類個體對酒精的敏感度差異極大。為了避免意外的發生，本研究將以老鼠取代人體作為急性酒精暴露的實驗對象，探討急性酒精暴露對巨噬細胞數目及免疫功能的影響，以作為人類酒精中毒者和酒癮者之免疫治療參考模式。酒精巨噬細胞神經內分泌物質ethanolmacrophageneuroendocrine mediatorsProject