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研究生:許華偉
研究生(外文):Hua-Wei Hsu
論文名稱:在老鼠模式中急性酒精暴露對腹腔巨噬細胞的影響
論文名稱(外文):Effects of ethanol on the peritoneal macrophages in a mouse model for binge drinking
指導教授:吳文俊吳文俊引用關係
指導教授(外文):Wen-Jun Wu
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:130
中文關鍵詞:酒精腹腔巨噬細胞細胞凋亡糖皮質素糖皮質素受器拮抗劑
外文關鍵詞:ethanolperitoneal macrophagesapoptosisglucocorticoidsRU486
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先前已有研究指出慢性酒精中毒者和酒精成癮者比非酗酒者較容易罹患傳染病及癌症,原因在於酒精會抑制免疫功能,因而增加感染的機會。巨噬細胞是聯結第一道先天性免疫防線和第二道特異性免疫防線的主角,在對抗外來的感染上佔有舉足輕重的地位。本篇研究以B6C3F1母鼠為實驗動物, 32 %(6.0 g/kg)的酒精劑量為實驗劑量,探討在急性酒精曝露下,對腹腔內巨噬細胞的細胞數及其免疫功能,包括吞噬作用、釋放過氧化氫及一氧化氮能力和毒殺腫瘤細胞能力的影響。研究結果發現酒精會減少腹腔巨噬細胞的細胞數及抑制上述所列之免疫功能。另外加入thioglycollate及polyinosinic-polycytidylic acid (poly I:C) 模擬生物體受到細菌及病毒感染時的情形,也發現酒精會降低腹腔巨噬細胞數及其免疫功能。我們也使用RU486(糖皮質素受器的拮抗劑)探討糖皮質素(glucocorticoids)對急性酒精降低腹腔巨噬細胞的細胞數及抑制免疫功能所扮演的角色,結果顯示酒精誘導生成的糖皮質素的確會在這方面對其造成影響。另外我們發現酒精會誘發腹腔巨噬細胞發生凋亡(apoptosis)導致腹腔巨噬細胞數的下降。在本研究中我們也發現急性酒精誘發生成的糖皮質素會同時啟動 bax - caspase-9 - caspase-3及FasL/Fas - caspase-8 - caspase-3這兩條細胞凋亡路徑。另外我們利用RU486及Fas突變老鼠或anti-FasL 抗體的方式證實糖皮質素和Fas的過度表現可大部份解釋經由酒精所引起的巨噬細胞凋亡。
Both clinical and laboratory reports indicate that ethanol (EtOH) addicts are prone to infections because EtOH can suppress the immue functions. Macrophages play an important role in host resistance to microbes and tumor cells. In this study, we investigated the effects of EtOH on the murine peritoneal macrophage cellarity and immue functions after a single dose of EtOH by gavage. The immue functions of macrophages including phagocytosis, hydrogen peroxide (H2O2) production, nitric oxide (NO) release and tumoricidal activity. The results showed that EtOH decreased macrophage number and inhibited the above-mentioned immue functions in a time-dependent manner. In addition, we found the similar phenomena of EtOH on both thioglycollate-elicited and polyinosinic-polycytidylic acid (poly I:C)-activated macrophages. We used RU486 (glucocorticoid receptor antagonist) to explore the role of EtOH in decreasing macrophage number and inhibiting macrophage functions. The result indicated that EtOH induced the production of glucocorticoids that were involved in this process. Furthermore, EtOH enhanced apoptosis of macrophages in a time-dependent manner. Also, we found that EtOH-induced production of glucocorticoids can trigger both bax - caspase-9 - caspase-3 and FasL/Fas - caspase-8 - caspase-3 apoptotic pathways to promote macrophage apoptosis. Finally, we demonstrated that glucocorticoids and Fas overexepression promoted the greater part of EtOH-induced macrophage apoptosis by administrating RU486 in Fas mutated mice or anti-FasL antibody treated mice.
目錄
壹、 中文摘要 1
貳、 英文摘要 3
參、 前言 5
肆、 材料與方法
一、 材料
1. 試劑 22
2. 實驗動物 23
二、 腹腔巨噬細胞的功能分析
1. 腹腔巨噬細胞製備 23
2. 巨噬細胞吞噬能力之分析 24
3. 巨噬細胞釋放一氧化氮能力之分析 25
4. 巨噬細胞釋放過氧化氫能力之分析 25
5. 巨噬細胞毒殺腫瘤細胞能力之分析 26
三、 DAPI染色法分析凋亡的巨噬細胞數目 27
四、 流式細胞分析儀(flow cytometry)分析凋亡 27
的巨噬細胞數目
五、 流式細胞分析儀測定腹腔內巨噬細胞的分布百分比28
六、 蛋白質樣品製備及定量分析
1. 蛋白質樣品製備 28
2. 蛋白質定量分析 29
七、西方墨點法(Western blot) 29
八、 結果分析 31
伍、 實驗結果
一、 急性酒精暴露對老鼠腹腔巨噬細胞數目及功能 32
的影響
二、 糖皮質素(glucocorticoids)在急性酒精暴露下對43
老鼠腹腔巨噬細胞數目及功能方面所扮演的角色
三、 急性酒精暴露對老鼠腹腔巨噬細胞發生細胞凋亡 50
的影響
四、 糖皮質素在急性酒精誘導腹腔巨噬細胞發生 62
細胞凋亡所扮演的角色
陸、 討論 81
柒、 圖表目錄
圖一、急性酒精暴露對老鼠腹腔內常駐的(resident) 90
巨噬細胞數目及功能的影響
圖二、急性酒精暴露對老鼠腹腔內發炎的(inflammatory) 91
巨噬細胞數目及功能的影響
圖三、急性酒精暴露對老鼠腹腔內激發的(primed) 92
巨噬細胞數目及功能的影響
圖四、比較餵食酒精與皮下注射corticosterone之 93
血中糖皮質素濃度
圖五、糖皮質素對老鼠腹腔巨噬細胞數目及功能的影響 94
圖六、急性酒精暴露對老鼠腹腔巨噬細胞發生細胞凋亡 96
的影響
圖七、急性酒精暴露對老鼠腹腔巨噬細胞發生細胞凋亡 97
的影響
圖八、Mitochondrial-dependent pathway在 98
急性酒精誘導老鼠腹腔巨噬細胞發生細胞凋亡
的影響
圖九、Mitochondrial-dependent pathway在 99
急性酒精誘導老鼠腹腔巨噬細胞發生細胞凋亡
的影響
圖十、FasL和Fas在急性酒精誘導老鼠腹腔巨噬細胞 100
發生細胞凋亡的影響
圖十一、急性酒精誘發生成的糖皮質素對老鼠腹腔 102
巨噬細胞發生細胞凋亡的影響
圖十二、急性酒精誘發生成的糖皮質素對老鼠腹腔巨噬103 細胞發生細胞凋亡的影響
圖十三、急性酒精誘發生成的糖皮質素促使老鼠腹腔內104
巨噬細胞走向mitochondrial -dependent pathway
圖十四、急性酒精誘發生成的糖皮質素對Fas- 106 dependent pathway誘導老鼠腹腔巨噬細胞發生細胞凋亡的影響
圖十五、比較MRL/MpJ 和B6C3F1品系其腹腔巨噬細胞 108
對酒精敏感性的差異
圖十六、比較MRL/MpJ 和B6C3F1品系其腹腔巨噬細胞 109
對酒精敏感性的差異
圖十七、Fas突變對急性酒精所誘發的糖皮質素促進 110
巨噬細胞發生細胞凋亡的影響
圖十八、Fas突變對急性酒精所誘發的糖皮質素促進 111
巨噬細胞發生細胞凋亡的影響
圖十九、阻斷 FasL對急性酒精所誘發的糖皮質素促進113
巨噬細胞發生細胞凋亡的影響
圖二十、阻斷 FasL對急性酒精所誘發的糖皮質素促進114
巨噬細胞發生細胞凋亡的影響
捌、 附表
附表一 115
附表二 116
玖、 參考文獻 117
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周碧瑟 台灣地區在校青少年藥物使用流行病學調查研究
(行政院衛生署八十八年度委託研究計畫 )
國人吸煙、喝酒、嚼檳榔及上下班使用交通工具之盛行狀況分析。
行政院衛生署檢疫總所疫情資訊。中華民國八十七年。
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