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研究生:紀惠美
研究生(外文):Hui-mei Chi
論文名稱:蜆水萃取液對正常與氧化傷害大白鼠初代肝細胞之保護作用
論文名稱(外文):Protective effects of water extract of clam on the normal and oxidants-treated primary rat hepatocytes
指導教授:周淑姿周淑姿引用關係
指導教授(外文):Su-tze Chou
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2005
畢業學年度:94
語文別:中文
論文頁數:81
中文關鍵詞:四氯化碳蜆水萃取液過氧化氫大白鼠初代肝細胞
外文關鍵詞:carbon tetrachloridewater extract of clamhydrogen peroxideprimary rat hepatocyte
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本實驗以蜆水萃取液(water extract of clam, WEC)為實驗材料,以大鼠初代肝細胞之分離與培養為實驗模式,探討WEC在各種濃度及不同時間培養下對肝細胞中麩胱甘(glutathione, GSH)相關生理活性之影響,以及四氯化碳或過氧化氫誘導肝細胞損傷後WEC對其保護作用。八週齡雄性Sprague-Dawley大鼠以二階段肝灌注技術進行大鼠初代肝細胞之分離與培養。肝細胞以WEC處理24或48小時後,分析細胞生存力、TBARS值、細胞內麩胱甘含量與麩胱甘相關酵素活性。結果顯示,以WEC培養肝細胞24或48小時後,各處理組之乳酸去氫滲漏率(lactate dehydrogenase leakage, LDH leakage)及TBARS值與控制組相比,皆無顯著性差異(P<0.05),故WEC之添加不會造成初代肝細胞損傷及增加細胞內脂質過氧化作用。WEC 0.84 mg/mL培養肝細胞24或48小時後,肝細胞內GSH含量與GSH/GSSG比值有顯著增加之作用(P<0.05)。另外,WEC之添加對麩胱甘硫轉移(glutathione S-transferase, GST) 、麩胱甘過氧化(glutathione reductase, GRd)及麩胱甘還原(glutathione reductase, GRd)活性有增加之趨勢,並於48小時培養後其增加趨勢更為顯著。肝細胞以不同濃度WEC預培養24小時後,再以四氯化碳或過氧化氫誘導肝細胞損傷之實驗結果顯示,四氯化碳(5 mM)或過氧化氫(10 mM)會顯著增加初代肝細胞乳酸去氫滲漏率,並減少細胞內GSH含量;當以WEC 0.84 mg/mL處理後,最能顯著減緩乳酸去氫滲漏率的增加及GSH含量的減少。另外,以WEC處理後能顯著減緩四氯化碳或過氧化氫所誘導之GST與GPx活性的下降。同時,WEC也能顯著增加過氧化氫所誘導細胞損傷後細胞內GRd活性(P<0.05)。綜合上述,WEC可經由增加肝細胞內GSH含量及GSH相關抗氧化及解毒代謝酵素活性,而提升肝細胞生存力、抗氧化及解毒代謝能力而達到保護細胞之作用。
The objective of this study was to investigate the effects of various concentrations and incubation time intervals of water extract of clam (WEC) on glutathione and its antioxidant and detoxication defense systems in the normal and oxidants-treated damaged primary rat hepatocytes. Two steps of liver perfusion combined with cell culture have been used in this study. The primary rat hepatocytes were treated with various concentrations of WEC for 24 or 48hr, and the cell viability, TBARS, intracellular glutathione (GSH) content and GSH-related enzyme activities were measured. Results showed that hepatocytes treated with 0.42-6.72 mg/mL WEC for either 24 or 48 hr,did not change LDH leakage and lipid peroxidation as compared with the controls (P&lt;0.05). In addition, 0.84 mg/mL WEC significantly increased the intracellular GSH content and GSH/GSSG ratio (P&lt;0.05). However, pretreatment of WEC increased the activities of glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GRd) after 24 and 48hr incubation.
In second experiment, the primary rat hepatocytes were pretreated with various concentrations of WEC for 24hr, followed by treating with 5 mM carbon tetrachloride (CCl4) or 10 mM hydrogen peroxide (H2O2) for an additional 1hr. Data showed CCl4 or H2O2 significantly increased the LDH leakages and decreased intracellular GSH content and GST, GPx and GRd activities as compared with the controls (P&lt;0.05). However, pretreatment of WEC (0.84 mg/mL) significantly increased the viability , GSH content, and the activities of GST and GPx as compared with the cells treated with CCl4 or H2O2 alone (P&lt;0.05). Additionally, the GRd activity was increased significantly by treatment of WEC as compared with the H2O2 alone (P&lt;0.05).
In conclusion, our results showed that WEC elevated the viability and the antioxidation and detoxication capabilities of hepatocytes by increasing the GSH level and the activites of GSH-related enzymes in hepatocytes.
綱 目..................... I
圖(表)次................. VI
中文摘要................... VIII
英文摘要................... X
第一章 前言............... 1
第二章 文獻回顧............ 4
第一節 台灣蜆.............. 4
一、蜆之分類與形態 .........4
二、台灣蜆之養殖........... 4
三、台灣蜆之加工利用....... 5
四、台灣蜆之營養組成份..... 5
五、蜆之保健功效........... 6
第二節 肝臟的生理功能與肝臟疾病之形成........ 8
一、肝臟生理功能............................. 8
二、肝病......................................9
第三節 自由基................................ 10
一、自由基的種類............................. 10
二、自由基的生成途徑..........................12
三、自由基與肝疾病的相關性................... 13
第四節 細胞內抗氧化防禦系統 ..................14
一、酵素性抗氧化系統......................... 15
二、非酵素性抗氧化系統....................... 16
第五節 四氯化碳誘導肝損傷之作用.............. 16
一、四氯化碳之毒性........................... 16
二、四氯化碳誘發肝損傷之機制................. 17
三、四氯化碳對抗氧化物質之影響............... 17
第六節 過氧化氫誘發肝損傷之機制.............. 18
第三章 實驗材料與方法........................ 20
第一節 實驗材料............................. 20
一、蜆水萃取液製備方法....................... 20
二、大鼠初代肝細胞之培養..................... 20
第二節 實驗方法............................. 22
一、大鼠初代肝細胞之分離與培養............... 22
二、樣品處理................................. 23
三、細胞存活率之分析......................... 24
四、脂質過氧化測定........................... 25
五、麩胱甘(glutathione, GSH)含量之分析.... 26
六、麩胱甘過氧化 (glutathione peroxidase, GPx)活性之分析.................................................... 27
七、麩胱甘還原 (glutathione reductase, GRd)活性之分析.....................................................28
八、麩胱甘硫轉移 (glutathione-S-transferase, GST)活性之分析.................................................. 28
九、蛋白質定量分析.................................... 28
十、統計分析.......................................... 29
第四章 結果........................................... 30
第一節 不同濃度蜆水萃取液(water extract of clam, WEC)對正常大鼠初代肝細胞生理活性之影響.......................... 30
一、不同濃度蜆水萃取液對正常大鼠初代肝細胞生存力之影響.................................................... 30
二、不同濃度蜆水萃取液對正常大鼠初代肝細胞脂質過氧化之影響.....................................................30
三、不同濃度蜆水萃取液對正常大鼠初代肝細胞內麩胱甘含量之影響.................................................. 31
四、不同濃度蜆水萃取液對正常大鼠初代肝細胞內GST、GPx與GRd活性之影響.............................................. 32
第二節 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳(carbon tetrachloride, CCl4)對肝細胞氧化損傷之影響.... 33
一、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對細胞生存力之影響........................................ 33
二、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對細胞內麩胱甘含量之影響................................ 33
三、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對細胞內GST、GPx與GRd活性之影響........................... 34
第三節 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫 (hydrogen peroxide, H2O2) 對肝細胞氧化損傷之影響...... 35
一、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫對細胞生存力之影響........................................ 35
二、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫對細胞內麩胱甘含量之影響................................ 35
三、以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫對細胞內GST、GPx與GRd活性之影響 ............................36
第五章 討論.......................................... 37
第一節 不同濃度蜆水萃取液對正常大鼠初代肝細胞生理活性之影響 ...............................................37
第二節 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對肝細胞氧化損傷之影響.................................. 40
第三節 以不同濃度蜆水萃取液預處理大鼠初代肝細胞損後過氧化氫對肝細胞氧化損傷之影響................................ 43
第六章 結論.......................................... 46
參考文獻.............................................. 63
&lt;附錄一&gt; 麩胱甘-抗氧化及解毒防禦系統................ 74
&lt;附錄二&gt; 四氯化碳代謝途徑............................. 75
&lt;附錄三&gt; 自由基造成細胞傷害之機制..................... 76
&lt;附錄四&gt; 不同濃度蜆水萃取液對大鼠初肝細胞中GST, GPx與GRd活性之影響.............................................. ......77
&lt;附錄五&gt; 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對肝細胞中乳酸去氫滲率之影響...........................78
&lt;附錄六&gt; 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫對肝細胞中乳酸去氫滲率之影響...........................79
&lt;附錄七&gt; 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後四氯化碳對細胞內麩胱甘含量之影響...............................80
&lt;附錄八&gt; 以不同濃度蜆水萃取液預處理大鼠初代肝細胞後過氧化氫對細胞內麩胱甘含量之影響...............................81
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