臺灣博碩士論文加值系統

已知飲酒會使肝臟受到慢性刺激引起持續性的傷害與修復過程，反覆的肝損傷最後導致肝臟纖維化。肝纖維化及發展最後階段的肝硬化已成為全世界主要的健康問題之一。相當多的證據提出肝星狀細胞活化為肝纖維化形成的中心環節。肝星狀細胞是肝纖維化時生成膠原蛋白的主要來源細胞；最近研究顯示肝星狀細胞也是金屬蛋白酶的主要來源，金屬蛋白酶參與基質修復並能分解基質。
而柴胡是已被拿來治療肝臟疾病的傳統中草藥，柴胡皂苷a、柴胡皂苷c及柴胡皂苷d為柴胡主要的有效成份。已知道柴胡皂苷在不同病理機制具治療的效果，例如抗發炎、抗菌與抗癌的功效。
本研究討論微米及奈米顆粒大小的柴胡甲醇萃取液對被乙醇刺激肝星狀細胞的抗肝纖維化效用。微米及奈米柴胡是分別經由傳統研磨及氣流崩散的奈米技術製成，並利用掃瞄式電子顯微鏡觀察柴胡外觀形態與顆粒大小。在萃取有效成份方面，柴胡甲醇萃取液經索式萃取方式萃取，再利用HPLC分析其有效成份。在細胞實驗模式中，柴胡萃取液加入肝星狀細胞作用後再以乙醇處理兩小時，利用MTT試驗及RT-PCR分析得到結果。
本研究顯示柴胡經奈米化技術處理後能更快速萃取出有效成份並且所萃取的有效成份含量比微米柴胡高。
結果顯示187.5mg/mL及250mg/mL奈米化柴胡的抗纖維化功效比187.5mg/mL及250mg/mL微米柴胡佳，可觀察到第一型膠原蛋白、第三型膠原蛋白及組織金屬蛋白酵素抑制子-1的mRNA隨著柴胡劑量增加而降低其表現量；基質金屬蛋白酶-2及基質金屬蛋白酶-13的mRNA隨著柴胡劑量增加而增加其表現量，顯示柴胡對酒精性肝纖維化有預防作用。

Alcoholic beverages are known to cause persistent wound-healing process of liver responses to a chronic stimulation. The repeated liver injury leads to liver fibrosis consequently. Liver fibrosis and its end stage sequela—cirrhosis become a major worldwide health problem. Considerable evidences suggest that the activated hepatic stellate cells (HSCs) play a key role in the fibrotic process. They are the major cells to synthesize the interstitial collagens, which characterize fibrosis. Recent works suggest that HSCs are also a source of matrix degrading metalloproteinase (MMPs), which could participate in matric remodeling.
The roots of Bupleurum Chinense DC. have been used in traditional Chinses herbal medicine for curing liver diseases. Saikosaponin-a, Saikosaponin-c and Saikosaponin-d are triterpene saponin derived from the roots of Bupleurum Chinense DC. Previous findings showed that Saikosaponins exhibit a variety of pharmacological and immunomodulatory activities including anti-inflammatory, anti-bacterial, antiviral and anti-cancer effects.
In this study, we investigate the anti-fibrosis effects of micro- Bupleurum and nano-Bupleurum on activated hepatic stellate cells stimulated via ethanol. The micro- Bupleurum and nano-Bupleurum were prepared by traditional grinder and pulsed air-flow pulverizer, respectively. The morphology of particles was examined by particle sizer and FE-SEM. For extracting the active component, Soxhlet extraction method was applied. The extracts were diluted to the same volume for HPLC analysis and exposed to the HSCs. At the indicated time, HSCs were exposed to ethanol for 2 hours and then analysize by MTT assay and RT-PCR.
The results showed that the nano-Bupleurum extracts revealed stronger anti-fibrosis bioactivities. The nano-Bupleurum extracts decreased the mRNA expression level of collagen type I, collagen type III and TIMP-1. On the contrary, they increased the level of MMP-2 and MMP-13. To conclude, the bioactive components of the 187.5mg/mL and 250mg/mL nano-Bupleurum released faster than the 187.5mg/mL and 250mg/mL micro- Bupleurum. Furthermore, the extracts of Bupleurum can alleviate HSCs injury from ethanol.

摘要 I
ABSTRACT ...................................................................................................... III
圖目錄 VII
表目錄 VIII
第一章 緒論 1
1.1 研究背景 ................................................................................................. 1
1.2 酒精性肝臟纖維化 ................................................................................. 1
1.3 肝纖維化病理機轉簡介 ......................................................................... 4
1.4 傳統中藥治療肝纖維化的研究 ............................................................. 6
1.5 奈米技術 ................................................................................................. 9
1.6 目的 ....................................................................................................... 12
第二章 材料與方法 ........................................................................................... 13
2.1 實驗設計 ............................................................................................... 13
2.2 實驗細胞 ............................................................................................... 13
2.3 柴胡甲醇萃取溶液的製備 ................................................................... 14
2.3.1. 細胞實驗的柴胡甲醇萃取溶液的製備 ........................................... 15
2.3.2. 不同萃取時間的柴胡甲醇萃取溶液的製備 ................................... 15
2.4 粒徑分佈檢測(Dynamic light scattering system, DLS) ....................... 16
2.5 掃瞄式電子顯微鏡 (Scanning electron microscopy, SEM) ............... 16
2.6 高效率液相層析(High Performance Liquid Chromatography, HPLC)
17
2.6.1 標準品之製備 .................................................................................... 17
2.6.2 HPLC 分析條件 ................................................................................ 17
2.7 MTT 呈色分析法 ................................................................................. 18
2.8 total RNA 的萃取 .................................................................................. 19
2.9 反轉錄反應（Reverse transcription; RT） .......................................... 21
2.10 半定量反轉錄聚合酵素連鎖反應 ....................................................... 21
2.11 統計方法 ............................................................................................... 22
第三章 結果 24
3.1 粒徑分析與掃瞄式電子顯微鏡的物性分析 ....................................... 24
3.2 柴胡有效成份分析 ............................................................................... 25
3.2.1. 細胞實驗的柴胡甲醇萃取溶液 ........................................................ 26
3.2.2. 不同萃取時間的柴胡甲醇萃取溶液的製備 ................................... 26
3.3 柴胡不同作用時間對HSC 活性試驗 ................................................. 30
3.4 細胞形態 ............................................................................................... 33
3.5 微米與奈米化柴胡對乙醇刺激HSC-T6 mRNA 表現之影響 ........... 33
第四章 討論 41
4.1 奈米化柴胡外觀型態 ................................................................................ 41
4.2 柴胡有效成份萃取 ................................................................................... 41
4.3 抑制肝星狀細胞纖維化 ........................................................................... 42
第五章 結論 43
經由奈米化技術處裡的柴胡，確實能夠達到快速萃取並且能夠萃取出更多
的有效成份。 ..................................................................................................... 43
第六章 參考文獻 ............................................................................................... 44


圖目錄
圖一 肝纖維化造成肝竇內細胞改變 ................................................... 5
圖二 實驗架構 ....................................................................................13
圖三 細胞實驗流程 ............................................................................14
圖四 細胞實驗流程 ............................................................................15
圖五 奈米化柴胡粒徑分布圖 ............................................................24
圖六 SEM 下奈米化柴胡SEM 下顆粒外觀形態及大小 ..................25
圖七 柴胡皂苷HPLC 層析圖 ............................................................28
圖八 不同萃取時間的柴胡皂苷a 含量 .............................................29
圖九 不同萃取時間的柴胡皂苷c 含量 .............................................29
圖十 不同萃取時間的柴胡皂苷d 含量 .............................................30
圖十一 柴胡作用1 小時 ....................................................................31
圖十二 柴胡作用3 小時 ....................................................................32
圖十三 柴胡作用6 小時 ....................................................................32
圖十四 細胞形態 ................................................................................36
圖十五 mRNA expression of collgen type I ........................................37
圖十六 mRNA expression of collgen type III .....................................37
圖十七 mRNA expression of MMP-2 .................................................38
圖十八 mRNA expression of MMP-13 ...............................................38
圖十九 mRNA expression of TGF-beta1 ............................................39
圖二十 mRNA expression of TIMP-1 .................................................39
圖二十一 mRNA expression of alpha-SMA .......................................40
表目錄
表一 柴胡移動相之梯度沖提 ............................................................18
表二 Primer sequences for PCR ..........................................................23
表三 柴胡標準品分析 ........................................................................27
表四 微米與奈米化柴胡有效成份濃度 .............................................27

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