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系統識別號 U0026-0812200915361352
論文名稱(中文) 以金奈米粒子及胸腺素α1治療四氯化碳誘發肝纖維化小鼠之研究
論文名稱(英文) Therapeutic Effects of Gold Nanoparticles and Thymosin α1 on CCl4-Induced Liver Fibrosis in Mice
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 王誌愷
研究生(英文) Chih-kai Wang
學號 s1694403
學位類別 碩士
語文別 中文
論文頁數 50頁
口試委員 口試委員-陳毓宏
指導教授-吳昭良
口試委員-蕭璦莉
口試委員-謝嘉興
口試委員-彭國証
中文關鍵字 胸腺素α1  肝纖維化  金奈米 
英文關鍵字 Liver Fibrosis  Thymosin α1  Gold Nanoparticles 
學科別分類
中文摘要 肝纖維化是肝臟受到慢性損傷時產生之反覆傷口修復反應所造成的結果。在肝纖維化過程中,肝星狀細胞 (hepatic stellate cell, HSC)被活化常是重要關鍵;而轉化生長因子-β1 (transforming growth factor-β1,TGF-β1)的分泌會促使HSC轉型為肌纖維母細胞 (myofibroblast),並刺激細胞外間質(extracellular matrix, ECM)的合成和抑制其降解,如此可使肝臟內增加許多纖維組織。利用四氯化碳(CCl4)誘導實驗動物肝損傷、肝纖維化及肝硬化是最廣泛應用於評估藥物是否具有抗纖維化之療效。胸腺素 α1(Thymosinα1, Tα1)是胸腺所產生的多種肽類物質之一,其序列為前胸腺素αprothymosinαProTα的前28個胺基酸所組成,它具有增強T細胞功能和調節免疫平衡等作用。因此在本研究中,我們利用四氯化碳誘發小鼠肝纖維化動物模式探討金奈米粒子及胸腺素α1抗肝纖維化之療效。小鼠每週兩次皮下注射四氯化碳,連續注射八週,並同時在隔天給予腹腔1.4 ml 之10 nM金奈米粒子及20 g 和glutathione S transferase (GST) 融合之T1蛋白,連續注射八週。治療完後,將小鼠犧牲,取其血清並檢測其天門冬胺酸轉胺酶 (aspartate aminotransferase, AST)及丙氨酸轉胺酶 (alanine aminotranferease, ALT)等肝功能指數,並測定乙型轉型生長因子(transforming growth factor-β1, TGF-β1)的含量,肝組織亦進行hematoxylin & eosin 和Masson-Goldner stain的病理變化檢測,並以西方墨點法(western blot)分析其磷酸化Smad2的表現量。我們的研究結果顯示,金奈米和Tα1治療可以顯著降低AST和ALT活性。根據H&E和Masson-Goldner染色分析,也發現金奈米和Tα1治療可以降低肝纖維化程度。金奈米和Tα1治療亦可降低TGF-β1的訊息傳遞。因此我們推測金奈米在四氯化碳誘發小鼠肝纖維化的狀態下,具有抗纖維化的效果,可能是透過與TGF-β1結合而阻斷其下游訊息傳遞;而Tα1在四氯化碳誘發小鼠肝纖維化的狀態下,可能是透過降低發炎反應而減少TGF-β1的表現來達到抗纖維化的效果。由此實驗可知,金奈米和Tα1治療利用不同的機制來達到抗纖維化的效果,也可提供肝纖維化新的治療途徑,並具有臨床應用的潛力。
英文摘要 Hepatic fibrosis is a process of wound healing in response to chronic liver injury. In the liver, activated hepatic stellate cells (HSCs) are the key mediators of fibrosis. Transforming growth factor-β1 (TGF-β1) induces the phenotypic transition of HSCs into myofibroblast cells, which enhance production of extracellular matrix (ECM) and attenuate the degradation of ECM. CCl4-induced liver damage, liver fibrosis, and liver cirrhosis in experimental animals have been used for assessing anti-fibrotic drugs. Thymosin α1 (Tα1) is a peptide produced by thymus and a 28 amino acid peptide derived by cleavage of prothymosinα(ProTα). It can augment T cell function and has immunomodulatory activities. In this study, we evaluated the anti-fibrotic effects of gold nanoparticles and Tα1 using a CCl4-induced liver fibrosis mouse model. Mice were injected subcutaneously with CCl4 twice a week for 8 weeks, and on the following day, 1.4 ml of 10 nM gold nanoparticles and 20 μg of glutathione S transferase (GST)-Tα1 fusion protein were giving intraperitoneally. All mice were sacrificed after 8 weeks of treatment. Their sera were assessed for the levels of aspartate aminotransferase (AST) and alanine aminotranferease (ALT) for monitoring hepatic function and for the content of TGF-β1. Liver tissues were also histopathologically examined with hematoxylin & eosin and Masson-Goldner stains. Moreover, the expression of phosphorylated Smad2 in the liver tissues was also detected by western blot analysis. We found that gold nanoparticles and Tα1 significantly reduced the levels of serum AST and ALT and the degree of liver fibrosis. Our results also showed that gold nanoparticles and Tα1 could reduce the signal transduction of TGF-β1 in the liver. These data suggest that gold nanoparticles may bind to TGF-β1 and block its downstream signaling pathway and that Tα1 may reduce inflammatory responses, resulting in reducing the expression of TGF-β1. These activities may have contributed to their anti-fibrotic effects. Collectively, our results suggest that gold nanoparticles and Tα1 exhibit anti-fibrotic effects by different mechanisms, which may be novel therapeutic approaches for liver fibrosis with potential future clinical applications.
論文目次 中文摘要 ………………………………………………………………………… I
Abstract …………………………………………………………………………III
致謝 …………………………………………………………………………… V
目錄 ……………………………………………………………………………VI
圖表目錄…………………………………………………………………………… IX
緒論(Introduction)……………………………………………………………… 1
肝纖維化簡介 ……………………………………………………………………1
肝纖維化病理機轉 ………………………………………………………………1
TGF- β1 ………………………………………………………………………… 2
TGF- β1與肝纖維化 ……………………………………………………………2
CCl4致肝纖維化方式 ………………………………………………………… 2
金奈米的應用 ………………………………………………………………… 3
胸腺素 α1(Thymosin α1,Tα1) …………………………………………… 3
研究動機與實驗設計…………………………………………………………… 4
材料與方法(Materials and Methods)………………………………………… 5
實驗動物 ……………………………………………………………………… 5
金奈米的合成…………………………………………………………………… 5
蛋白質表現 …………………………………………………………………… 5
蛋白質純化 …………………………………………………………………… 6
蛋白質電泳(SDS-PAGE)……………………………………………………… 7
肝纖維化動物模式 …………………………………………………………… 8
小鼠血清和組織的採集………………………………………………………… 9
血清生化學分析………………………………………………………………… 9
組織病理學評估…………………………………………………………………11
Masson-Goldner染色及評分方式…………………………………………… 11
酵素免疫分析法 (Enzyme-linked immunosorbent assay,ELISA) ………12
西方墨點法 (Western blot analysis)………………………………………… 13
統計分析 ……………………………………………………………………… 14
Part 1 …………………………………………………………………………… 15
實驗結果(Results)……………………………………………………………… 16
肝纖維化模型的建立………………………………………………………… 16
金奈米可以改善肝功能AST和ALT……………………………………… 16
以H&E染色法分析金奈米對肝纖維化病理狀況之改善情形……………… 16
以Masson-Goldner染色法分析金奈米對減少膠原纖維在肝內沈積的情形 ………………………………………………………………………………17
以TGF-β1 ELISA和pSmad2 Western blot來分析金奈米減少TGF-β1訊息傳遞的情形 …………………………………………………………………… 17
討論(Discussion) …………………………………………………………… 18
參考文獻(References)……………………………………………………… 20
Part 2 …………………………………………………………………………… 26
實驗結果(Results)……………………………………………………………… 27
GST fusion protein的純化………………………………………………… 27
肝纖維化模型的建立 ……………………………………………………… 27
Tα1可以改善肝功能AST和ALT………………………………………… 27
以H&E染色法分析Tα1對肝纖維化病理狀況之改善情形 ……………… 28
以Masson-Goldner染色法分析Tα1對減少膠原纖維在肝內沈積的情形 …………………………………………………………………………… 28
以TGF-β1 ELISA和pSmad2 Western blot來分析Tα1減少TGF-β1訊息傳遞的情形 …………………………………………………………………… 28
討論(Discussion) ……………………………………………………………… 30
參考文獻(References) ………………………………………………………… 31
圖表(Figures)…………………………………………………………………… 37
自述 …………………………………………………………………………… 50
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