臺灣博碩士論文加值系統

在本篇論文中，我們以源自人類肝細胞株-Hep3B為實驗模式，並以Transforming growth factor- beta1 (TGF- beta1)來引發細胞的凋亡作用(apoptosis)，以此來探討龍膽瀉肝湯的作用。此外，龍膽瀉肝湯的五種主藥成分，即gentiopicroside, baicalein, geniposide, alisol B monoacetate及saikosaponin-d (分別來自龍膽草，黃芩，梔子，澤瀉及柴胡)，其對於Hep3B細胞的作用也加以深入探討。首先我們以形態的鑑定及annexin V的螢光標定確認TGF- beta1所引發的細胞死亡為凋亡作用。龍膽瀉肝湯以濃度相依性抑制了TGF- beta1所引發的凋亡作用；進一步分析其主要成分的作用，發現gentiopicroside及baicalein也能有效的抑制此凋亡作用，但geniposide卻沒有抑制作用；然而，alisol B monoacetate及saikosaponin-d本身即對Hep 3B細胞造成死亡作用。Alisol B monoacetate是造成細胞凋亡作用，而saikosaponin-d則是造成細胞壞死(necrosis)。此外，我們也探求了caspase-3的酵素活性，由實驗結果得知，TGF- beta1會明顯的增加caspase-3的活性，gentiopicroside及baicalein都能有效的抑制caspase-3的活性，而geniposide則沒有任何抑制作用；經由更進一步的分析細胞凋亡與Caspase-3的活性發現，細胞凋亡的程度與caspase-3的活性成正相關，其相關係數r值為0.94，可見caspase-3的活性調控在TGF- beta1引發細胞凋亡及中藥成分抑制凋亡的機轉中，扮演了一個很重要的角色。在上述的主成分中，baicalein是效果最好的成分，因此，我們將baicalein的作用做一詳細的探討。在此，我們以一些藥理作用劑，例如a-hydroxyfarnesyl phosphoric acid、PD098059以及wortmannin都不能有效的抑制baicalein的作用，暗示著baicalein抗凋亡作用並不經由Ras/Raf-1/MAPK及PI 3-kinase的相關路徑。然而，很有趣的一點是baicalein及baicalein加上TGF- b1都能明顯的增加內生性H2O2的生成，再則，TGF- beta1會明顯降低細胞NF-kB的nuclear translocation，而baicalein本身便能很明顯的增加NF-kB的nuclear translocation，進而消除掉TGF- beta1在這方面的作用。由此看出NF-kB的nuclear translocation在這凋亡/抗凋亡的作用扮演了非常重要的角色。因此，綜合以上所述，龍膽瀉肝湯對於TGF- beta1所引起的細胞凋亡有保護作用，而其中baicalein是最有效的主要成分，且baicalein之抗細胞凋亡作用主要是增加NF-kB的nuclear translocation。

In the present study, we have found that Long-dan-tan (Chinese name), one of the most common traditional herbal drugs in Chinese people with chronic liver diseases, exhibits the inhibitory effect on TGF-beta1-induced apoptosis in human liver-derived cell line Hep 3B. We examined the antiapoptotic action of Long-dan-tan and its five major ingredients, such as gentiopicroside, baicalein, geniposide, alisol B monoacetate and saikosaponin-d. Firstly, we confirmed the TGF-beta1-induced apoptosis by cell morphology identification and in situ labeling of annexin V-reaction techniques. Long-dan-tan mixture showed a concentration- dependent inhibition on TGF-beta1-mediated apoptosis. Furthermore, the ingredients, gentiopicroside and baicalein, but not geniposide, effectively prevented TGF-beta1-induced apoptosis. By contrast, alisol B monoacetate and saikosaponin-d themselves without TGF-beta1 caused the cytotoxicity in Hep 3B cells. We then determined the caspase-3 activity in the present study. It showed that TGF-beta1-induced a profound increase in caspase-3 activity; gentiopicroside and baicalein, but not geniposide, had a significant suppression on this TGF-beta1-induced caspase-3 activity. To further correlate the functional apoptosis and caspase-3 activity, a good coefficient (r value) of 0.94 was obtained suggesting the central role of the regulation of caspase-3 activity in TGF-beta1-induced effect and the antiapoptotic action of these herbal ingredients. Among these ingredients, baicalein showed the most effective one in the present work. The detailed investigation was therefore carried out. Several pharmacological agents, such as a-hydroxyfarnesyl- phosphoric acid, PD098059, and wortmannin, were examined to have little influence on baicalein-mediated antiapoptotic effect suggesting baicalein action is independent of the Ras/Raf-1/MAPK- and PI3-kinase pathways. Interestingly, baicalein alone as well as baicalein plus TGF-beta1 significantly stimulated the formation of H2O2. Furthermore, baicalein alone profoundly induced the nuclear translocation of NF-kB. On the contrary, TGF-beta1 almost completely suppressed this translocation suggesting the crucial role of NF-kB translocation on the apoptotic/antiapoptotic regulation. We conclude that Long-dan-tan has a protective effect on TGF-beta1-induced cell apoptosis. Baicalein is the most effective ingredient among Long-dan-tan mixture. Furthermore, baicalein exerts the antiapoptotic action via activation of NF-kB nuclear translocation.

縮寫表------------------------------1
中文摘要----------------------------3
英文摘要----------------------------5
第一章 研究動機與目的---------------7
第二章 文獻回顧---------------------9
第三章 實驗材料與方法
一、實驗材料-----------------30
二、細胞培養-----------------31
三、實驗方法-----------------31
第四章 結果
第一部份---------------------38
第二部分---------------------42
第五章 討論
第一部份---------------------48
第二部分---------------------54
第六章 結論------------------------60
圖表-------------------------------61
參考文獻---------------------------83

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