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| 題名: | 傳統中藥方劑一貫煎加味在肝纖維化的作用與機轉
The effect and mechanism of Modified Yi Guan Jian, A Chinese herbal decoction on Liver Fibrosis |
| 作者: | 林宏任;Hung-Jen Lin |
| 貢獻者: | 中醫學院中醫學系博士班 |
| 關鍵詞: | 肝纖維化;肝星狀細胞;細胞凋亡;粒線體路徑;liver fibrosis;hepatic stellate cell;cell apoptosis;mitochondria pathway |
| 日期: | 2010 |
| 上傳時間: | 2010-09-29 12:00:38 (UTC+8) |
| 摘要: | 肝硬化是常見的慢性、進行性、瀰漫性肝病，由一種或數種病因長期或反覆作用引起。在肝細胞受損過程中，肝的星狀細胞受到刺激而被活化，進而形成過度的肌纖維母細胞樣細胞堆積，導致肝纖維化之形成。
本研究目的，欲證明中醫臨床依辨證後，所使用的方劑一貫煎加味用於治療肝纖維化後其分子作用及機轉；在HSC-T6細胞株實驗中以MTS 分析法觀察其增殖情形，以TUNEL 分析法觀察其凋亡，以西方墨點法檢測α-SMA、Bcl-2、Bax、cytochrome c、caspase-3/-9。以二甲基亞硝胺老鼠動物造模4周後以本方劑口服2周，觀察其血清生化、病理組織學，計算 SSS 分數，檢測Hydroxyproline，以RT-PCR觀察α-SMA、Collagen Ⅰ、TIMP-1之m RNA表達，也利用西方墨點法檢測α-SMA蛋白質之表現。
實驗研究結果顯示：(1)本方劑對HSC-T6細胞株有讓其細胞有凋亡之作用。(2) 凋亡路徑經由ROS 蓄積而誘發粒線體的內在路徑，引起其釋放鈣離子及cytochrome c至胞質中，也使抗凋亡家族Bcl-2的下調，凋亡家族的Bax上調，最後活化了caspase-9 及caspase-3此細胞凋亡路徑。(3)本實驗經DMN誘發老鼠肝纖維化後以本方劑治療2周，結果發現α-SMA無論在mRNA基因及蛋白質的表達都有下降現象。(4)檢測肝纖維化的黃金指標：Hydroxyproline也明顯降低。以SSS 計分法亦得到相同結果。在生化功能檢查中也明顯下降，肝功能轉好，顯示本方劑對肝臟有保護作用。(5)細胞外基質抑制降解因子- TIMP-1，其mRNA 之表達也明顯降低，顯示一貫煎加味對膠原降解的活性增加，故本方劑具有降解纖維化能力，明顯減輕肝纖維化。
Liver cirrhosis is a common chronic, progressive hepatic disease. When liver damaged, the hepatic stellate cells would be activated and leading to over accumulation of the myofibroblast-like cells. The excessive deposition of extracellular matrix (ECM) is causing to liver fibrosis.
The purpose of our study is to prove the effect and mechanism of Chinese medicine -modified Yi Guan Jian on liver fibrosis. During the HSC-T6 (cell line) experiment, we observed cell growth with MTS assay and cell apoptosis with TUNEL assay and also tested α-SMA, Bcl-2, Bax, cytochrome c, caspase-3,-9, with Western Blot. We also established fibrosis animal model in rats with dimethylnitrosamine. After 4 weeks those animals of the herbal group were fed modified Yi Guan Jian for 2 weeks and then their biochemical blood serum, pathology and histology were observed. The SSS points were also calculated, tested with Hydroxyproline, and to observe the mRNA expression of α-SMA, Collagen Ⅰ, and TIMP-1 with RT-PCR. The expression of α-SMA protein with western blot was also observed.
Finally our study concluded that:
1) The prescription induced cell apoptosis with HSC-T6 cell line.
2) The apoptosis pathway went through ROS and caused a mitochondria pathway which released the calcium ion and cytochrome c into cytosol and also decreased the anti-apoptotic family Bcl-2 and upregulated the apoptotic family Bax, and lastly it activated the apoptosis pathway of caspase-9 and caspase-3.
3) In this study after inducing liver fibrosis with DMN to treat rats for 2 weeks with this prescription, the results show the mRNA expression of α-SMA and protein decreased.
4) To test the gold index of liver fibrosis: Hydroxyproline obviously decreased and also the same result of SSS score was found, the biochemistry function decreased obviously and the liver fibrosis improved. That means this prescription can protect the liver.
5) Extracellular matrix (ECM) inhibitor: TIMP-1 the expression of mRNA obviously decreased. It shows modified Yi Guan Jian could reduce the collagen content which means this prescription is able to decrease the liver fibrosis and greatly improve the situation in rats. |
| 顯示於類別: | [中醫學系暨碩博班] 博碩士論文
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