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研究生:林敬甄
研究生(外文):Jing-Jen Lin
論文名稱:小球藻 (Chlorella sorokiniana) 對酒精性肝損傷之保護作用-以吳郭魚為動物模式
論文名稱(外文):Protective effects on liver damage of Chlorella sorokiniana using tilapia as an alternative animal model
指導教授:孫寶年孫寶年引用關係
指導教授(外文):Bonnie Sun Pan
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:96
中文關鍵詞:吳郭魚小球藻
外文關鍵詞:tilapiaChlorella sorokiniana
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吳郭魚作為模式動物優點包含具有細胞試驗缺乏的代謝機制,且相較於囓齒動物便宜,具潛力成為細胞試驗接續囓齒動物間的動物模式。故本研究以吳郭魚建立肝纖維化之動物模式,初步篩選保健食品護肝之成分。
吳郭魚以酒精透過腹腔注射誘導損傷,其酒精濃度以 20 % 最佳,此濃度不易造成吳郭魚死亡,也不會使吳郭魚不進食。經每 2 天腹腔注射 0.4 ml, 6 週後吳郭魚的比成長率 (specific growth rate, SGR) 下降、死亡率及肝體比 hepatosomatic index (HSI) 上升,並且肝指數丙胺酸轉胺酶 (alanine aminotransferase, ALT) 上升,血液抗氧化力下降,經組織切片觀察,可發現肝細胞內堆積三酸甘油酯,形成脂肪細胞的型態,肝臟中三酸甘油酯與總膽固醇提高,肝臟更有血球浸潤作用,並可活化肝星狀細胞,使肝臟中的維生素 A 含量下降。
透過此肝損傷動物模式探討小球藻 (Chlorella sorokiniana) 是否具有護肝之功效。飼料中分別添加 5 % 及 10 % 小球藻,餵食 6 週,確定可以顯著改善 SGR、HIS 及肝損傷 (降低血漿中 ALT 的活性)。此外,小球藻可使肝臟中的三酸甘油酯、總膽固醇及維生素 A 的含量恢復至正常值。另外,攝食小球藻可增加血漿中葉黃素之含量。

由此,吳郭魚腹腔注射酒精與嚙齒動物以酒精液態飼糧誘導形成之脂肪肝症狀相似,且小球藻在此動物模式中具有預防肝損傷之功效。

The purpose of this study was to develop tilapia as a liver damage alternative animal model for screening bioactivity compound.
The advantage of using tilapia as an animal model is having the metabolism which cell culture model doesn’t have. Tilapia may serve as a practical compromise animal model between the cell culture models and the rodent models, which is relatively labor-intensive and expensive.
Tilapia was intraperitoneal injection of ethanol to induce liver damage. The optimal concentration of injected ethanol was 20 %, which won’t cause tilapia anorexia or death. After intraperitoneal injection of 20 % ethanol 0.4 ml per two days for 6 weeks, specific growth rate (SGR) reduced, mortality rate, hepatosomatic index (HSI) and liver function index-alanine aminotransferase (ALT) increased and the antioxidant capacity in blood was reduced. The morphology of hepatocytes were accumulated with triacylglycerol to form as fat cell, and the liver tissue had red blood cells infiltration. Moreover, the concentration of triacylglycerol and total cholesterol in liver was increased. Hepatic stellate cells (HSC) seemed to be activate, thus the contents of retinol in liver was reduced.
Using tilapia liver damage animal model to evaluate the protective effect of Chlorella sorokiniana. Tilapia was intraperitoneal injection of ethanol and fed with diet containing 5 % or 10 % Chlorella sorokiniana for 6 weeks. Chlorella sorokiniana caused significant amelioration of SGR, HSI and liver damage (reduced the activity of plasma ALT). Moreover, Chlorella sorokiniana partially improved the concentration of triacylglycerol, total cholesterol and retinol in liver returned to normal values. In addition, Chlorella sorokiniana could increase the concentration of lutein in plasma.
These results demonstrate that tilapia intraperitoneal injection of ethanol could induce fatty liver symptom was similar to rodent animal models induced by ethanol diet, and Chlorella sorokiniana may have the effect of prevention of hepatic damage in tilapia liver damage animal model.

中文摘要 i
英文摘要 iii
壹、前言 1
貳、文獻整理 3
一、小球藻之生態 3
二、小球藻之生理活性 4
1. 抗氧化 4
2. 降血糖 4
3. 降血脂及膽固醇 5
4. 降血壓 5
5. 排除外生物質 (xenobiotic) 6
6. 調節免疫 7
7. 抗發炎 8
8. 抗腫瘤 8
三、肝纖維化 (FIBROSIS) 8
1.誘發因子 9
1.1 病毒感染 9
1.2 酒精性脂肪肝 10
1.3 非酒精性脂肪肝炎 11
2.病理機轉 12
2.1 氧化壓力 12
2.2 發炎反應 13
2.3 肝星狀細胞 (hepatic stellate cells, hsc) 之活化 15
四、肝損傷動物模式 16
五、吳郭魚對酒精之代謝作用 18
参、實驗架構 19
1.酒精致死濃度試驗 19
2.酒精誘導肝損傷試驗 19
3.小球藻投餵吳郭魚經酒精誘導肝損傷試驗 20
肆、材料與方法 21
一、材料 21
1.純種莫三比克吳郭魚(Oreochromis mossambicus) 21
2.雜交吳郭魚(Oreochromis niloticus × Oreochromis aurea) 21
3.小球藻 (Chlorella sorokiniana) 21
二、方法 22
1.一般成分分析 22
2.建立以酒精誘導吳郭魚肝纖維化之動物模式試驗 22
2.1確定酒精劑量之預備試驗 22
2.2 吳郭魚酒精肝損傷試驗 23
2.3小球藻投餵酒精誘導吳郭魚肝纖維化養殖試驗 24
3. 採血及血漿製備 24
4. 肝臟切取與肝臟上清液製備 25
5. 蛋白質定量 25
6. 一般生理指標 26
6.1 肥滿度(condition factor, K) 26
6.2 比成長率 (specific growth rate, SGR) 26
6.3 肝體比 (hepatosomatic index, HSI) 26
6.4 死亡率 26
7. 抗氧化力測定 27
8.ALT、AST 酵素活性測定 27
9. 三酸甘油酯 (triacylglycerol, TG)定量 28
10. 總膽固醇 (total cholesterol) 定量 29
11. 血漿中 HDL 與 LDL 29
12. 脂質過氧化 (thiobarbituric acid-reactive substances, TBARS)測定 30
13. α-tocopherol、lutein、retinoid 定量 30
13.1 自血漿萃取之方法 30
13.2 自肝臟萃取之方法 31
13.3 HPLC 分析條件 31
14. 肝臟中膽固醇及三酸甘油酯定量 32
15. LOXs 活性測定 32
16. 肝臟組織切片 33
16.1 石蠟切片 33
16.2 hematoxylin and eosin (h&e) 染色 34
16.3 masson's trichrome 染色 34
17. 肝細胞的影像判定 35
18. 統計分析 35
伍、結果與討論 36
一、酒精濃度對吳郭魚致死之影響 36
二、注射酒精對吳郭魚之影響 39
1. 肝指數 39
2. 血脂濃度 41
3. 肝臟組織型態 42
三、小球藻對酒精誘導吳郭魚脂肪肝之影響 46
三、小球藻對酒精誘導吳郭魚脂肪肝之影響 46
1. 一般生理變化 46
2. 血液之變化 49
2.1 肝指數及血脂濃度 49
2.2血液抗氧化力 53
3. 肝臟之變化 55
3.1 組織型態 55
3.2 總膽固醇及三酸甘油酯含量 64
3.3脂質過氧化之變化 66
3.4 脂氧合酶活性 68
4. 血漿及肝臟中葉黃素及維生素 A、E之變化 70
4.1 葉黃素 70
4.2 維生素 A 73
4.3 維生素 E 75
四、吳郭魚與老鼠酒精性肝損傷動物模式之比較 77
五、小球藻劑量對吳郭魚酒精性肝損傷之影響 79
陸、結論 83
柒、參考文獻 85


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