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研究生(外文):Mei-Chih Liao
論文名稱(外文):Inhibitory Effects on TGF-β-induced Apoptosis by Rhubarb in Hep3B/T2 Cells 
指導教授(外文):Sheau-Farn Yeh
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肝癌是世界上非常普遍的惡性疾病之一,然而不同的肝臟疾病如病毒肝炎、肝纖維化及肝硬化均可能惡化並轉變成肝癌。許多肝臟疾病的病理機制皆可能是因細胞凋亡失去調節,過度的細胞凋亡的確會導致嚴重的肝臟損傷。轉型生長因子(TGF-□)常會大量表現於患有肝臟疾病甚至是肝癌的病人身上,TGF-□會在肝細胞中引起不當的細胞凋亡並使之癌化。前人研究結果表明TGF-□可在人類肝癌Hep3B/T2細胞中引起細胞凋亡。TGF-□主要是藉由Smad訊息傳遞路徑誘發apoptotic protein,或藉大量產生ROS,接著活化一連串的caspases以促使細胞凋亡。 因此,本論文將以Hep3B/T2細胞探討如何防止細胞免於TGF-□引起的細胞凋亡。
中草藥自古以來便認為具有多方面的療效,因此成為許多研究者的焦點。在本論文中利用Cell Death Detecton ELISA與流式細胞儀證明中草藥大黃酒精萃取物能隨著濃度提高而抑制TGF-□誘發Hep3B/T2細胞凋亡,因此希望能更進一步探討大黃之抑制作用點;我們分別以caspase活性分析及免疫雜化法發現大黃可降低TGF-□活化caspases以及下游基因如CTGF表現,表示大黃成功抑制了TGF-□誘發細胞凋亡並且具有防止肝纖維化的潛力。研究並以流式細胞儀偵測細胞內TGF-□誘發ROS的相對產生量,結果顯示大黃可遏止ROS的產生甚至具有清除ROS的能力。此外又以Luciferase為報導基因測得大黃會降低TGF-□□response elements活性,間接證明大黃可干擾 TGF-□引起的 Smad訊息路徑。但觀察MAPK protein kinase(ERK)大黃無法回復TGF-□所造成的phosphoERK suppression,也許大黃抑制TGF-□誘發Hep3B/T2細胞凋亡著力點非在此。本研究亦嘗試純化分離大黃可抑制TGF-□誘發細胞凋亡的有效成份。
Hepatocellular carcinoma (HCC) is one of the common malignant diseases worldwide. However, there are many potential risks like viral hepatitis B and C, liver fibrosis, and liver cirrhosis that may contribute to the pathogenesis of HCC. Dysregulation of apoptosis is a principal mechanism in many liver diseases. Indeed, excessive apoptosis can lead to severe liver damage. Transforming growth factor □□□(TGF-□)□ overexpression is frequently observed in liver disease even in HCC patients. TGF-□□may induce abnormal apoptosis in hepatocytes and extend pathogenesis. Previously studies showed that TGF-□ could induce apoptosis in human hepatoma Hep3B/T2 cells. It illustrated that the TGF-□-induced apoptotic pathway were through Smad signaling to express apoptotic proteins and production of reactive oxygen species followed by activation the caspase cascades. In this thesis, we studied the mechanism of rescue TGF-□-induced apoptotic in Hep3B/T2 cells using Chinese herb Rhubarb.
A lot of different aspects have been utilized in Chinese herbs, however, these herbs effect are not clear yet for some specific disease. Therefore it recently is great interested by the researchers in study the Chinese herb.
In this thesis, we found that Chinese herb Rhubarb inhibited TGF-□-induced apoptosis in a dose-dependent manner using Cell Death Detection ELISA and Flow Cytometry in cultured Hep3B/T2 cells. We showed that Rhubarb inhibited TGF-□-induced apoptosis on suppressing initial or effectors caspases, TGF-□ downstream gene expression (ex. CTGF), and ROS production by examine the activity of caspases, or measuring the ROS production thru flow cytometry and immune hybridization of specific antibodies. Furthermore, Rhubarb inhibited TGF-□-induced apoptosis thru Smad signaling pathway by transiently transfected 3TP-Lux containing TGF-□ response elements. However, Rhubarb did not reverse TGF-□-suppressed phosphor-ERK, a protein related to cell proliferation. Purification and characterization the mechanism of the active principles from Rhubarb on the rescue of TGF-□-induced apoptosis was also undergoing.
Taken together, this study demonstrated that Rhubarb prevented TGF-□-induced apoptosis through suppression of TGF-□-induced Smad signaling and ROS production. This study provides a new route to explore the therapeutic strategies for liver disease like liver fibrosis and liver cirrhosis.
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