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研究生:羅琬瑜
研究生(外文):Wan Yu Lo
論文名稱:腎上腺皮質醇與性類固醇激素對吳郭魚非特異性免疫之影響:吞噬作用
論文名稱(外文):The Effects of Corticosteroids and Sex steroids on Non-Specific Immunity in Tilapia:Phagocytosis
指導教授:張清風張清風引用關係
指導教授(外文):Ching Fong Chang
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:86
中文關鍵詞:腎上腺皮質醇性類固醇非特異性免疫吞噬作用吳郭魚
外文關鍵詞:CorticosteroidsSex steroidsNon-Specific ImmunityPhagocytosisTilapia
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本論文研究之目的在探討腎上腺皮質醇(corticosteroids)與性類固醇(sex steroids)對吳郭魚與鯉魚循環系統中吞噬細胞調控的作用及機制。實驗主要分三大部分: (1) 吳郭魚的離體(In vitro)細胞培養實驗 (2) 吳郭魚的活體(In vivo)實驗 (3) 鯉魚的離體細胞培養實驗。
以五種濃度(1pM、0.1nM、10nM、1μM及0.1mM / well)的各種corticosteroids與sex steroids進行4小時的吳郭魚白血球細胞培養後,其中cortisol、dexamethasone、triamcinolone acetonide、cortisone、aldosterone、estradiol-17β(E2)及ethynylestradiol有明顯抑制吞噬作用之能力,且有dose dependent的關係,但estrone、androgens、progesterone及T3則無顯著之效果。在cortisol與E2的共同作用下,對吞噬活性之抑制效果有加成的現象,顯示這兩種激素的作用途徑並不相同,各具有特異性的receptors。再以不同濃度蛋白質轉錄抑制劑actinomycin D及轉譯抑制劑cycloheximide分別與cortisol及E2參與4小時細胞培養,得知此兩種抑制劑對cortisol與E2有拮抗之反應。顯示這兩種激素對吳郭魚白血球吞噬活性的抑制作用是經由與特異性的glucocorticoid receptors及E2 receptors接合後,進入胞內而影響細胞之基因表現所造成。
將在25℃馴化下之吳郭魚施予急速低溫處理,降溫至12℃及13℃。在維持12℃達30分鐘及13℃達2小時的實驗中,均顯示吞噬活性並無受到立即的影響,卻在1-2天後開始下降,至第4天左右血液中的白血球吞噬能力降至最低,而在往後幾天又逐漸恢復。另外加入注射cortisol之實驗組,顯示注射的濃度越高,對吞噬作用的抑制能力越強,與離體實驗相似,且施予急速低溫緊迫加上注射高劑量(2.5mg/100g B.W.) cortisol之實驗魚其吞噬能力復原情形顯然低於其他組別。
鯉魚離體細胞培養實驗中,只有同屬於corticosteroids的cortisol與dexamethasone對吞噬活性有顯著抑制的作用且抑制效果不強,顯示不同魚種之白血球對激素的感受性也應該有所差異。
The objectives of the study were to investigate the regulation of corticosteriods and sex steriods on the phagocytosis in tilapia and common carp. The peripherial blood leucocytes culture system was developed for the phagocytosis experiment. The study was divided into three parts, 1) phagocytosis in vitro in tilapia, 2) phagocytosis in vivo in tilapia, 3) phagocytosis in vitro in common carp.
Five doses (1pM、0.1nM、10nM、1μM及0.1mM / well) of corticosteroids and sex steroids were conducted in the expriments. Cortisol, dexamethasone, triamcinolon acetonide, cortisone, aldosterone, estradiol-17βand ethylestradiol could significantly surppress phagocytosis activity in a dose-dependent manner in tilapia after 4 hours incubation. However, cortisone, aldosterone, estrone, testosterone, 11-ketotestosterone and progesterone only had less significant or no any effects on phagocytosis in tilapia. Actinomycin D and cycloheximide could block the supression effects of cortisol and estradiol on the phagocytosis in tilapia. The data suggest the cortisol and estradiol regulates phagocytosis in tilapia leucocytes through glucocorticoid receptors and estrogen receptors, respectively.
Acute cold stress had a significant effects on decreasing phagocytosis in tilapia. The lowest phagocytosis activity was observed after 4 days of cold stress. Exogenous cortisol (in vivo) also could suppress phagocytosis activity in tilapia. The phagocytosis activity of common carp only could be slightly affected by cortisol and dexamethasone but not in other steroids. The species-specific difference was suggested on the regulation of phagocytosis in teleosts.
中文摘要………………………………………………………………………I
英文摘要…………………………………………………………………….III
圖目錄………………………………………………………………………VIII
壹、前言………………………………………………………………………1
貳、材料與方法………………………………………………………………12
一、實驗用魚…………………………………………………………………12
二、抗凝血劑與培養液的製備………………………………………………12
三、分離吳郭魚與鯉魚之白血球……………………………………………13
四、螢光乳膠珠液與quenching buffer之製備……………………………14
五、激素及其他藥品之配製…………………………………………………14
六、分析方法…………………………………………………………………16
七、Giemsa染色法……………………………………………………………17
八、實驗設計…………………………………………………………………17
(一)探討吳郭魚白血球進行吞噬作用的最佳吞噬物比例、培養時間及
quenching time……………………..………………………………………17
(二)各種激素離體實驗對吳郭魚吞噬作用之影響…………………………18
(三)急速低溫對吳郭魚PBLs吞噬作用之影響………………………………20
(四)急速低溫及注射不同劑量cortisol對吳郭魚PBLs吞噬作用與cortisol對曾遭受緊迫吳郭魚PBLs吞噬作用之影響…………………………………21
(五)激素在離體實驗(In vitro)對鯉魚PBLs吞噬作用之影響……………23
九、統計分析…………………………………………………………………24
參、結果………………………………………………………………………25
一、 利用Giemsa染色法觀察吞噬細胞……………………………………25
二、 探討激素與低溫處理對吳郭魚及鯉魚吞噬作用之影響……………25
(一) 探討進行吳郭魚白血球的最佳吞噬物比例、培養時間及quenching time……………………………………………………………………………25
(二)各種激素在離體實驗對吳郭魚PBLs吞噬作用之影響…………………26
(三)急速低溫對吳郭魚PBLs吞噬作用之影響………………………………29
(四)急速低溫及注射不同劑量cortisol對吳郭魚PBLs吞噬作用與cortisol對曾遭受緊迫吳郭魚PBLs吞噬作用之影響…………………………………30
(五)激素在離體實驗(In vitro)對鯉魚PBLs吞噬作用之影響……………31
肆、討論………………………………………………………………………33
伍、結論……………………………………………………………………..40
陸、參考文獻…………………………………………………………………41
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