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系統識別號 U0026-0812200914101353
論文名稱(中文) 以A酸衍生物分子抑制TGF-β所誘導早期肝臟纖維化的研究
論文名稱(英文) Using retinoic acid derivative molecules to antagonize the TGF-beta induced early liver fibrosis
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 96
學期 2
出版年 97
研究生(中文) 楊昆霖
研究生(英文) Kun-Lin Yang
電子信箱 s5889103@ccmail.ncku.edu.tw
學號 s5889103
學位類別 博士
語文別 中文
論文頁數 79頁
口試委員 召集委員-鄭瑞棠
口試委員-戴謙
口試委員-張定宗
指導教授-李益謙
口試委員-陳啟楨
口試委員-黎煥耀
中文關鍵字 轉型生長因子  水動力方式  早期肝臟纖維化  星狀細胞  A酸衍生物 
英文關鍵字 Transformation growth factor-β  Hydrodynamics-based  early liver fibrosis  retinoic acid derivative  Hepatic stellate cells (HSC) 
學科別分類
中文摘要 目前治療肝硬化唯一的方法就是肝移植,但因為捐贈者有限,限制了這項技術的應用。隨著肝纖維化的進展,尤其是在硬化階段,發病率與死亡率亦伴隨提高。因此,建立早期纖維化以研究如何預防纖維化於初期,是一非常重要的研究課題。過去一直認為抗自由基可以間接的抗纖維化,但在體內試驗一直令人失望。事實上,肝纖維化一旦進入臨床可發現的階段,其纖維化已經不可逆。很多研究肝纖維化的動物實驗,是以不同的藥物處理,如四氯化碳,誘導肝產生纖維化。這個方法雖然已被廣泛使用,但在這個模式,肝纖維化之程度未能清楚界定,很可能已變成不可逆。所以本研究的第一個策略,是建立早期肝臟纖維化的小鼠模式。有很多報告顯示轉型生長因子(tansformation growth factor-β, TGF-β),經由活性氧家族(reactive oxygen species;ROS)上調膠原蛋白1A2(Collagen 1A2;Col 1A2)基因的表現。這個過程是經由發炎反應(環氧酶-2;cycloxygease-2;Cox-2),導致星狀細胞活化,再促進纖維化。所以我們提出的第二個策略,是建立篩選平台;以ROS、COX-2與Col 1A2促進子,篩選小分子並評估其抗肝纖維化的效率。
我們利用水動力方式(hydrodynamics-based)將質體在5-7 秒內,經由尾靜脈注入小鼠體內,結果發現在同時餵與ZnSO4 的小鼠,TGF-β有最顯著的表現,其在血清中的含量,48 小時達到900-600 pg/ml 最高量,而其下游蛋白p-Smad2/3-Smad4,Sp1 也伴隨增加,而TGF-β、Sp1 在第五天恢復正常值。TGF-β為早期纖維化表現最顯著之細胞激素,且在本模式的表現,具可恢復性,我們因此認為此模式為早期纖維化小鼠模式。
我們以前述之篩選平台,從數十種藥物(小分子)篩選有效的標的,篩選到一個小分子,是從桑黃菌絲體萃取之A 酸衍生物(在本研究標誌為PL),並進而在早期纖維化的動物模式下,評估抗纖維化的效率。A 酸衍生物在30, 150, 300 nM 低濃度下,即能有效降低TGF-β在HSC-T6 細胞所誘導之ROS、胞內鈣離子之濃度、Cox-2 及Col 1A2 的表現,並能抑制Smad4、Smad2/3 轉位進入細胞核內。在動物模式下,評估hydroxyproline 及以RT-PCR 分析Col1A2 mRNA 的表現。此二項表現分別為控制組的50%與0.001%,並能改善ALT(alanine transaminase)及Sp1 轉錄蛋白的表現,且Masson’s 染色亦證實確實具有抗纖維化之效果。
結果顯示由桑黃萃取之A 酸衍生物,無論在體內或體外,皆能預防肝纖維化於早期,其機制可能經由阻止ROS、發炎反應與HSC 細胞的活化。我們的這個模式對肝有非常高之專一性且在臨床研究肝的早期纖維化,非常有效率。氧化性傷害為肝纖維化的病因之一。因此,以研發同時能抗氧化、抗發炎抗纖維化來預防肝纖維化之發生,為一重要而可行的策略;但此等小分子是否能進一步抗肝硬化或應用於臨床來治療肝纖維化或硬化,則需待後續的研究。
英文摘要 At present, the only curative treatment for end stage liver cirrhosis is organtransplantation. However, the availability of donor organ limits the application of this treatment. The current anti-fibrotic effect of antioxidants is disappointing in vivo due probably to the fact that once liver fibrosis is established it is too advanced to be reversed by medical intervention. Many studies of liver fibrosis consist of animal testing with various hepatotoxins such as carbon tetrachloride (CCl4). Although this method is often used, the model at which cirrhosis or extensive fibrosis becomes irreversible has not been well defined and is not represen-
tative of early-stage fibrogenesis. Therefore, it is of imperative importance to establish a suitable fibrosis model for studying the prevention of liver fibrosis at the initiation stage. This thesis investigation addresses this problem and sets out firstly to establish a different animal model for studying the early liver fibrosis.
It has been well documented that transforming growth factor-beta-1 (TGF-β1) up regulates the expression of α(1) collagen (Col 1A2) gene in the liver via the reactive
oxygen species (ROS). The process triggers inflammation (cycloxygenase-2; Cox-2), leading to hepatic stellate cells (HSC) damages and liver fibrogenesis. We hence screened small molecules isolated from some herbal medicines with the hope that they may antagonize fibrosis. We used ROS, Col 1A2 and Cox-2 promoter assays as screening targets. Out of a panel of approximately 96 chemical compounds for possible candidate that may inhibit liver fibrosis, a small retinoic acid molecule isolated from the mycelia of Phellinus linteus (denoted here as PL) was found to be a promising candidate molecule for inhibiting liver fibrogenesis when assayed by our newly established animal model where a plasmid containing TGF-β1 cDNA was“hydro-dynamically” transferred into an animal to generate a transient liver fibrosis.
The procedure involves a large-volume and high-speed intravenous injection of naked plasmid DNA into the animal tail vein; the whole procedure was finished within 5-7
seconds. We found that PL could strongly alleviate the expressions of hydroxyproline and Col 1A2 mRNA, making their expressions only about 50% and 0.001%,respectively, as compared to the pPK9a-transferred mice upon induction by ZnSO4. PL also markedly improves the expressions of alanine transaminase (ALT), Cox-2, α-SMA, p-Smad2/3 and trans- cription factor Sp1. Our results suggest that the retinoic acid derivative of Phellinus linteus prevents liver fibrosis from happening at an early phase both in vitro and in vivo through the inhibition of ROS, inflammation and the acti-
vation of HSC. Our model is more unique to the liver and may have its usefulness in clinical study for prevention of early stage liver fibrosis. As for its effects on advanced fibrosis or cirrhosis further studies are warranted.
論文目次 目錄…………………………………………………………… I
中文摘要……………………………………………………… IV
英文摘要 ………………………………………………… … VI
誌謝…………………………………………………………… VIII
圖表目錄…………………………………………………………IX
縮寫………………………………………………………………X
第一章 緒論…………………………………………………… 1
第一節 背景及重要性介紹…………………………………… 1
第二節 肝臟簡介……………………………………………… 1
第三節 正常肝臟與纖維化肝臟之比較……………………… 2
第四節 纖維化之機制………………………………………… 2
第五節 Transforming Growth Factor-β (TGF-β)介紹……3
第六節 活性氧化物(ROS)與肝纖維化……………………… 6
第七節 細胞凋亡在肝纖維化扮演之角色 …………………… 7
第八節 纖維化與癌症………………………………………… 7
第九節 抗纖維化與抗癌……………………………………… 8
第十節 基質金屬蛋白酵素
(matrix metalloproteinase, MM) ………………… 9
第十一節四氯化碳所造成之肝纖維化模式…………………… 9
第十二節早期肝臟纖維化小鼠模式建立之原理……………… 10
第十三節 小分子………………………………………………… 10
第二章 建立早期肝臟纖維化小鼠模式……………………… 12
第一節 緒論……………………………………………………… 12
第二節 實驗方法………………………………………………… 12
2-2.1 動物………………………………………… …………… 12
2-2.2 cDNA建構………………………………………………… 12
2-2.3 質體的純化與放大 ……………………………………… 12
2-2.4 水動力的轉染法……………………………………………12
2-2.5 血清中TGF-β的含量………………………………………13
2-2.6 誘導TGF-β1在肝臟的表現……………………………… 13
2-2.7 西方墨點分析………………………………………………13
2-2.8 羥脯氨酸含量………………………………………………14
2-2.9 血液生化分析 …………………………………………… 14
2-2.10 組織和免疫組織化學分析TGF-β1和α-SMA的表現……14
2-2.11 Masson三層染色………………………………………… 15
2-2.12 以反轉錄和即時PCR( Reverse transcription (RT)
and real time quantitative PCR)分析膠原蛋白
的表現………………………………………………… 15
2-2.13 統計分析………………………………………………… 16
第三節 結果討論………………………………………………… 16
2-3.1 TGF-β1基因在水動力轉植鼠之表現…………………… 16
2-3.2 肝臟巨觀之變化……………………………………………16
2-3.3 組織與免疫組織化學分析…………………………………17
2-3.4 血清生化分析………………………………………………17
2-3.5 膠原蛋白在轉植鼠的表現…………………………………17
2-3.6 膠電泳移動改變分析(Gel electrophoretic mobility
shift assays;EMSA) Sp1 蛋白…………………………17
2-3.7 討論…………………………………………………………18
第三章 小分子對抗TGF-β所誘導的早期肝臟纖維化………… 19
第一節 緒論……………………………………………………… 19
第二節 實驗方法………………………………………………… 19
3-2.1 HSC-T6細胞 ……………………………………………… 19
3-2.2 化學物 …………………………………………………… 19
3-2.3 cDNA 建構………………………………………………… 19
3-2.4 以流體細胞儀測量胞內ROS與α-SMA的表現…………… 20
3-2.5 轉染與雙螢光酶分析(Transfection and
dual-luciferase assay) ……………………………… 20
3-2.6 測量胞內鈣離子濃度的改變(cytosolic free
Ca2+;[Ca2+]i) ………………………………………… 21
3-2.7 細胞免疫螢光影像 ……………………………………… 21
3-2.8 p-Smad2/3, α-SMA 與Cox-2西方墨點分析…………… 21
第三節 結果與討論……………………………………………… 22
3-3.1 PL對胞內TGF-β所誘導之ROS之影響…………………… 22
3-3.2 PL對TGF-β所誘導之Col 1A2與Cox-2促進子之影響……22
3-3.3 PL降低HSC-T6細胞內鈣離子的濃度及α-SMA的表 …… 23
3-3.4 PL影響Smad4 和p-Smad2/3在HSC-T6細胞內分佈的位置 23
3-3.5 PL對以水動力轉送TGF-β轉植鼠所誘導膠原蛋白表現
之影響………………………………………………………24
3-3.6 血清之生化分析…… …………………………………… 24
3-3.7 西方墨點分析p-Smad2/3, α-SMA 和 Cox-2之
蛋白質表現量…………………………………………… 24
3-3.8 膠電泳移動改變分析…………………………………… 24
3-3.9 討論……………………………………………………… 25
第四章 討論……………………………………………………… 27
第五章 結論……………………………………………………… 29
參考文獻………………………………………………………… 30
附圖……………………………………………………………… 40
附錄……………………………………………………………… 58
論文發表……………………………………………………………79
附件(已發表之文)………………………………………………80
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