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| 題 名 | 利用基因微陣列圖譜與動物之離體及活體模式評估中藥ZC008抗肝臟纖維化之功效(2-1)=Microarray Profiling Delineates Molecular Portrait of the Anti-Fibrosis Effects of a Chinese Herbal Medicine, ZC008, by Using in Vivo and in Vitro Models (2-1) |
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| 作 者 | 徐士蘭; 黃奇英; | 書刊名 | 中醫藥年報 |
| 卷 期 | 29:8 2011.09[民100.09] |
| 頁 次 | 頁1-24 |
| 專 輯 | 中醫藥基因體相關研究 |
| 分類號 | 414.32 |
| 關鍵詞 | 肝硬化; 肝星狀細胞; 中草藥; 細胞凋亡; 基因微陣列; ZC008; Liver fibrosis; Hepatic stellate cell; Herbal medicine; Apoptosis; Microarray; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 研究目的:雖然醫療技術一直在進步,但台灣地區因罹患肝疾病而致死之人數仍然高居不下。在肝疾病中除肝癌外,最常發現的即為病毒性肝炎及肝硬化。而肝纖維化是各種慢性肝病進展為肝硬化的必經途徑, 其實質是肝內細胞外基質(Extracellular matrix)的合成與降解失去平衡,導致細胞外基質尤其是膠原蛋白的過渡沈積和分佈異常。隨著肝纖維化程度加重,異常增生的纖維束包纏形成假小葉,使正常肝臟的小葉結構和功能被破壞,即形成肝硬化。肝纖維化是可逆轉的,因此,抗肝纖維化治療是十分重要的。體內、外研究證實肝星狀細胞(Hepatic stellate cell)及其活化形式即轉變成纖維細胞是細胞外基質的主要來源,活化的肝星狀細胞,釋放TGF-β1 等多種細胞因子,從而促進細胞外基質的異常合成,抑制其降解。故肝星狀細胞的活化是肝纖維化形成過程的中心環節。據此,人們提出了針對形成肝星狀細胞活化的多個環節進行治療的策略。目前國內外有關肝纖維化治療的研究多處於實驗階段,尚乏有效療法。近年政府對中醫藥及天然藥物之研發工作日趨重視,也明訂中藥科學化與新藥研究開發列為國家重點發展項目。更選定中草藥為具有發展潛力之生物科技優先支持項目。本研究計畫是針對過去本研究室發現之具有治療肝硬化功效之中草藥代號ZC008,以培養之大鼠肝星狀細胞探討ZC008 抗肝纖維化作用於肝星狀細胞之分子機轉。期能藉由建立之離體細胞培養模式,給予中草藥科學化之驗證。 研究方法:本研究將以初代培養之大鼠肝星狀細胞為研究模式,以細胞數目測定、TUNEL 凋亡分析、Caspase 活性分析、西方點墨蛋白質分析、及免疫螢光染色分析等技術,探討肝星狀細胞處理ZC008 後其細胞的活化轉型之抑制及細胞凋亡等分子作用,藉此離體培養方式快速找到ZC008 具有治療肝硬化之分子作用機制。同時使用大鼠肝纖維化動物模式,結合基因微陣列分析。 結果與討論:結果發現,中草藥ZC008 在低濃度時具抑制肝星狀細胞生長之能力,而高濃度ZC008 處理可促進肝星狀細胞凋亡之作用。此ZC008 誘發之肝星狀細胞凋亡,是經由活化內生性的粒腺體致死路徑,因為活化了Caspase-3 及-9。同時ZC008處理肝星狀細胞可抑制alpha smooth muscle actin 及collagen I 分子的表現。肝臟中之星狀細胞被活化轉型而成為肌纖維化之細胞,此細胞大量在肝臟中增殖,破壞了正常肝組織的結構,使肝臟功能不足,導致患者死亡。因此若能抑制肝星狀 細胞的活化轉型及增生,即可有效的抑制肝纖維化,可抑防止肝硬化。目前國內外有關肝纖維化治療的研究雖然不少,但多處於實驗階段,尚乏有效療法。本研究探討已知有抗肝纖維化作用之中藥ZC008,是可藉由促進肝星狀細胞之凋亡,達到治療之效應。將大鼠肝纖維化動物模式,以現代化之科學技術,結合基因微陣列分析對此藥治療肝纖維化之功效給予科學化之驗證,此研究結果可使ZC008之醫藥價值受到國際醫界之認同。本研究計畫實驗模式之建立,亦可當作是中藥科學化平台架構之範本。 |
| 英文摘要 | Aim: Hepatic fibrosis, a precursor of cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver disease, and involves the abnormal accumulation of extracellular matrix (ECM), particularly collagens. It has been shown that the activation of stellate cells in injured livers leads to their proliferation and transformation into myofibroblast-like cells. The activated hepatic stellate cell is now well established as the key cellular element involved in the development of hepatic fibrosis. Herbal medicines that have been used in China for thousands of years are now being manufactured in China as drugs with standardized quality and quantity of ingredients. In the present study, the in vitro cultured rat hepatic stellate cells were used to examine the biological and molecular actions of ZC008(a selected herbal medicine which has been presently found by our laboratory to be the most safety and effective drug in treatment with chronic liver diseases, especially those with chronic hepatitis and liver fibrosis). The goal of this study is to evaluate the hepatoprotective effect of ZC008 on in vitro cultured hepatic stellate cells. Methods: Hepatic stellate cells from rat liver were treated with various concentrations of ZC008 extract for different duration. Cell death were assessed by direct cell number counting and TUNEL assay. Caspase activity was measured using specific fluorogenic substrates. The expression and cellular distribution of alpha smooth muscle actin and collagen I were examined by Western blotting and immunostaining. Dimethylnitrosamine (DMN)-induced liver fibrosis model was performed. Liver samples were immediately removed after sacrifice and prepared for histopathological staining. Blood samples, collected from the animals at autopsy, were used to measure serum concentrations or activity of biomarkers. Gene expression profilings were examined by Affymetrix GeneChip system (RG-U34A chip) and analysis by GeneSpring solfware 7.3. Results & Discussion: We found that ZC008 extract is the most potency drug among the tested drugs to treat of hepatic stellate cells. Treatment with ZC008 decreased the expression of alpha smooth muscle actin and collagen I in primary cultured stellate cells. DNA fragmentation and caspase activation were detected in ZC008-treated stellate cells, suggesting that ZC008 extract triggered a apoptotic cell death through a intrinsic mitochondrial pathway in this primary cultured hepatic stellate cells. The current results indicate that curing the liver fibrosis by ZC008 may be due to direct elimination of the activated fibrotic cells by the agent. This study intends to build a hypothesisdriven research on liver fibrosis at the system level by implementation of diverse research approaches with the goal to elucidate novel insights into the effect of ZC008 on liver fibrosis. We used dimethylnitrosamine (DMN) to induce rat necroinflammatory and hepatic fibrosis in a 6-week time course. Using the Affymetrix RG-U34A chip, 256 differentially expressed genes, including 44 necroinflammatory- related and 62 fibrosis-related, were demonstrated from the liver injury tissues. Moreover, ZC008, a Chinese herbal medicine, were validated to be an excellent anti-liver damage candidate drug by histopathological, biochemical and microarray analysis. Our proposed research model may provide a new exploratory modality for traditional Chinese medicine research. |
本系統中英文摘要資訊取自各篇刊載內容。