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研究生:劉勇男
研究生(外文):Yung-Nan Liu
論文名稱:以大白鼠胰臟轉分化肝細胞探討奈米薑黃素之肝化學毒性保護作用
論文名稱(外文):Chemoprotective Function of Nanoencapsulated Curcumin in Pancreatic AR42J Cell Transdifferentiated Hepatocyte
指導教授:龔瑞林龔瑞林引用關係沈家寧
指導教授(外文):Zwe-Ling KongChia-Ning Shen
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:2008
畢業學年度:96
語文別:中文
論文頁數:80
中文關鍵詞:奈米包覆轉分化薑黃素AR42J
外文關鍵詞:nanoencapsulationtransdifferentiationcurcuminAR42J
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薑黃素產自薑黃 (Curcuma longa Linn.) 地下莖,為一自然黃色色素,具有抗氧化、抗發炎、保肝等生理活性。近年研究指出,大白鼠胰臟癌細胞株 AR42J 細胞可以經由地米賽松 (dexamethasone)、抑瘤素 (oncostatin M, OSM)、胰島素等添加,誘導細胞轉分化成為類肝細胞。因此本研究利用此轉分化模式作為保肝食品篩選平台,確認四氯化碳對 AR42J 之細胞毒性影響,並以生物矽膠化法及矽膠化法製備成薑黃素奈米粒,探討薑黃素奈米粒之保肝效果。利用 real time RT-PCR 分析細胞轉分化後肝相關基因表現,其中 TAT (tyrosine amino- transferase) mRNA大量表現,在轉分化第 13 天基因表現與控制組相比達 6 倍以上。以 ELISA 偵測白蛋白含量,轉分化後相較於初代肝細胞約有 60% 製造白蛋白的能力。經由細胞存活率實驗後,使用 0.003 (v/v) 四氯化碳損傷 1 小時作為刺激條件。而薑黃素奈米粒之細胞存活率,IC50 約為 15~20 �慊/mL 且薑黃素奈米粒對 AR42J 細胞其毒性較薑黃素強,之後實驗以 15 �慊/mL 作為樣品劑量。先添加薑黃素奈米粒後,再與四氯化碳共培養,AR42J/H cells 之 細胞內 ROS 含量與AST、ALT 活性均顯著低於正控制組、薑黃素組別,且在人類肝癌細胞株 C3A cells 也有類似的效果。本研究結果顯示,AR42J 可轉分化成為類肝細胞。且確實薑黃素奈米粒對於四氯化碳所誘導類肝細胞損傷,有顯著預防的效果。
Curcumin, which is a natural yellow pigment. Product isolated from turmeric (Curcuma longa Linn.), has antioxidation, anti-inflammation, hepatoprotection activities. Pancreatic exocrine cell line AR42J can be transdifferentiated to hepatocytes by exposing it to dexamethasone, oncostatin M and insulin. In the currant study, we would like to establish a hepatoprotective screening platform and to investigate whether treatment can generate hepatotoxicity on transdifferentiation hepatocyte derive from AR42J cells. Furthermore, we examine the hepatoprotective effect by using different nanoencapsulated curcumin such as silicification and biosilicification on AR42J transdifferentiation hepatocyte (AR42J/H). The results showed that AR42J/H cells demonstrated several in vitro characters, including a 6 fold enhancement in related liver gene expression and a 60% albumin production capability compared with primary hepatocyte. In the part of hepatoprotective study, the hepatocytes, were subsequently treated with using 0.003 (v/v) CCl4 for 1 hour in AR42J/H as the injury condition. In MTT assay, curcumin nanoparticles had an IC50 of 15~20 �慊/mL, nanoencapsulated curcumin have substantial cytotoxicity. In ROS production, nanoencapsulated curcumin decreased the AST and ALT enzyme activity of AR42J/H, more than the positive control, silymarin and curcumin significantly. The results were similar in human hepatoma cell line C3A. In conclusion, nanoencapsulated curcumin enhance the hepatoprotective effect against CCl4-induced hepatotoxicity in transdifferentiated hepatocytes.
謝誌 i
Abbreviation iii
摘要 v
Abstract vi
目錄 viii
圖表目錄 xii
附錄目錄 xiv
壹、引言 1
貳、文獻回顧 3
一、肝臟病變 3
(一) 簡述肝相關疾病成因 3
(二) 四氯化碳誘導肝損傷 3
二、肝臟生理研究類型 3
(一) 動物模式 4
(二) 細胞模式 5
三、薑黃素 (Curcumin) 7
(一) 簡介薑黃素 7
(二) 薑黃素的物理及化學特性 7
(三) 薑黃素之生理功能 8
(四) 薑黃素的代謝 12
四、奈米科技應用 13
(一) 奈米科技簡介 13
(二) 奈米藥物載體 13
參、實驗設計 16
肆、實驗材料與方法 17
一、細胞種類與細胞培養 17
二、藥品、試劑 17
(一) 細胞培養 17
(二) 分化試劑 18
(三) Real-time RT-PCR 18
(四) ELISA 測定白蛋白分泌量 18
(五) 薑黃素奈米粒製備 18
(六) 細胞存活率測定 (MTT assay) 19
(七) 螢光染色法分析四氯化碳對細胞產生 ROS 19
(八) 天門冬胺酸轉氨? (aspartate transaminase, AST) 活性分析 19
(九) 丙胺酸轉氨? (alanine transaminase, ALT) 活性分析 19
三、實驗方法 19
(一) AR42J 細胞株轉分化實驗 19
(二) 即時反轉錄鏈鎖反應 (real-time RT-PCR) 19
(三) ELISA 測定白蛋白分泌量 20
(四) 四氯化碳誘導肝臟細胞損傷 21
(五) 薑黃素奈米粒製備 21
(六) Cell viability assay 22
(七) 利用螢光染色法分析四氯化碳對細胞產生 ROS 的影響 22
(八) 天門冬胺酸轉氨? (aspartate transaminase, AST) 活性分析 22
(九) 丙胺酸轉氨? (alanine transaminase, ALT) 活性分析 23
四、統計方法 23
伍、結果與討論 24
一、胰臟癌細胞株轉分化為類肝細胞之生理特性 24
(一) 轉分化時間對 AR42J 細胞形態的轉變 24
(二) 轉分化後對 AR42J 細胞中肝臟相關基因的表現 24
(三) 轉分化後對 AR42J 細胞分泌白蛋白的影響 25
二、四氯化碳在不同條件下對 AR42J/H 之細胞毒性 25
(一) 四氯化碳在不同劑量下對 AR42J/H 細胞毒性之影響 25
(二) 四氯化碳不同處理時間對 AR42J/H 細胞毒性之影響 25
三、薑黃素奈米粒對四氯化碳誘導損傷之保肝作用 26
(一) 薑黃素奈米粒之細胞毒性探討 26
(二) 利用螢光分析法分析薑黃素奈米粒抑制四氯化碳引起不同細 胞產生 ROS 之影響 27
(三) 薑黃素奈米粒抑制四氯化碳引起不同細胞之天門冬胺酸轉氨?釋出之影響 27
(四) 薑黃素奈米粒抑制四氯化碳引起不同細胞之丙胺酸轉氨?釋出之影響 28
陸、結論 30
柒、參考文獻 31
捌、圖表 42
玖、附錄 61
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