臺灣博碩士論文加值系統

肝纖維化是由於肝臟遭受到反覆性損傷後，所產生的一種對傷口修復之反應，過程中可見到肝臟細胞被活化並造成過量的細胞外基質蛋白之堆積。研究亦指出星狀細胞的活化和致纖維化是導致肝臟纖維化的主要病理機制。因此，抑制星狀細胞的活化及致纖維化，甚至誘使其走向細胞凋亡的途徑均被視為治療肝纖維化的策略之ㄧ。薑黃素，是來源於中藥薑黃 (Curcuma longa Linn) 的一種黃色色素，其具有抗發炎和抗氧化的作用。而轉型生長因子-β1 (Transforming growth factor-beta1)是一個具多功能的細胞激素，可促進肝臟纖維化的形成。本研究即利用不同濃度 (1~40 μM)的薑黃素來探討其在體外實驗裡對肝臟纖維化的作用及機轉。
實驗中，在加入或未加入薑黃素的條件下，以1 ng/ml 濃度的TGF-β1來刺激大鼠肝臟星狀細胞HSC-T6。濃度範圍1.25~10 μM 的薑黃素對於包括α-肌動蛋白 (α-SMA)、膠原蛋白及α-肌動蛋白之信使核糖核酸等纖維化指標物質的抑制性影響分別以西方墨點法、Sircol red assay和逆轉錄-聚合酶連鎖反應 (RT-PCR)來偵測。藥物對HSC-T6細胞存活率的影響由MTT分析法來測試。此外，濃度20~40 μM的薑黃素對於星狀細胞T6進行細胞凋亡的誘導作用則以Hoechst染色與propidium iodide染色之流式細胞儀來測定。然後我們也利用西方墨點法來探討薑黃素對細胞凋亡中細胞色素c (cytochrome c)自粒線體釋出到細胞質內的影響。
我們的結果顯示，1 ng/ml 濃度的TGF-β1可以有效地增加星狀細胞對膠原蛋白的製造 (達控制組的145 ± 5 %)，而在預先投予濃度範圍為1.25~10 μM的薑黃素後可對TGF-β1引起的刺激反應產生濃度效應的抑制作用；薑黃素在濃度10 μM下可明顯地抑制TGF-β1引起的膠原蛋白沉積 (達控制組的97 ± 7 %) 而不致產生細胞毒性。此外，1 ng/ml 濃度的TGF-β1亦可有效刺激細胞中α-肌動蛋白及其信使核糖核酸的表現，若預先投予同樣濃度範圍的薑黃素也可對此刺激作用產生濃度效應的抑制反應。另一方面，當單獨給予星狀細胞較高濃度範圍20~40 μM的薑黃素時，其可明顯並呈現濃度效應關係的見到細胞進行凋亡途徑，且過程中同時發現伴隨著細胞色素c由粒線體釋放至細胞質裡的現象，此亦存在著濃度效應關係。
因此我們認為薑黃素可能是依據著濃度的高低而展現其抗肝纖維化的兩種作用：即一為在低濃度時 (1.25~10 μM) 其具有抗致纖維化的作用，而在此範圍濃度下藥物並沒有明顯的細胞毒性；另一則是在高濃度時 (20~40 μM) 薑黃素又可藉由增加活化型星狀細胞粒線體內細胞色素c 釋放到細胞質而誘導細胞產生細胞凋亡的現象。由以上的實驗結果發現薑黃素具有治療或預防肝臟纖維化的潛力。

Hepatic fibrosis is a wound-healing response to repeated liver injuries and is characterized by the activation of hepatic stellate cells (HSCs) and excess accumulation of extracellular matrix (ECM) proteins. Activation and fibrogenesis of HSCs have been implicated in the pathogenesis of liver fibrosis. Therefore, suppression of activation and fibrogenesis of HSCs, as well as induction of apoptosis have been proposed as therapeutic strategies against liver fibrosis. Curcumin, a yellow curry pigment from turmeric (Curcuma longa Linn), has been shown to possess anti-inflammatory and antioxidant properties. Transforming growth factor-beta1 (TGF-β1) is a multifunctional growth factor and is the most potent cytokine for enhancing hepatic fibrogenesis. In this study, we investigated the in vitro effects of curcumin on hepatic fibrosis at the concentration range of (1~40 μM).
A cell line of rat HSC-T6 was stimulated with 1 ng/ml TGF-β1 in the presence or absence of curcumin. The inhibitory effects of curcumin (1.25~10 μM) on fibrosis markers including α-smooth muscle actin (α-SMA), collagen and the mRNA expression of α-SMA were assessed by immunoblotting, Sircol collagen assay and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The cell viability affected by curcumin was measured by MTT assay. In addition, the induction effects of curcumin (20~40 μM) on apoptosis in HSC-T6 cells were also determinated by Hoechst stains and flow cytometry analysis. Then we also investigated the release of cytochrome c from the mitochondria to the cytoplasm by using immunoblotting.
Our results revealed that TGF-β1 at 1 ng/ml induced collagen deposition of HSC-T6 cells (145 ± 5 % of controls), and pre-exposure of cells to curcumin (1.25~10 μM) inhibited the stimulatory effect of TGF-β1 in a concentration-dependent manner. Curcumin (10 μM) significantly reduced the TGF-β1-induced collagen deposition (97 ± 7 % of controls) without cytotoxicity in the MTT assay . In addition, TGF-β1 at 1 ng/ml also increased mRNA and protein expressions of α-SMA and pretreatment of curcumin (1.25~10 μM) concentration-dependently attenuated the up-regulating effect by TGF-β1. On the other hand, curcumin (20~40 μM) alone significantly and concentration- dependently enhanced apoptosis of HSC-T6 cells and this effect was accompanied by the induction of cytochrome c release from the mitochondria to the cytoplasm in a concentration-dependent manner.
We suggested that curcumin exerted anti-fibrotic effects, possibly through two different strategies depending on its concentrations. At lower concentrations (1.25~10 μM), curcumin exerted anti-fibrogenetic effect without causing significant cytotoxicity to HSC-T6 cells and at higher concentrations (20~40 μM), curcumin exerted induction of apoptosis in HSC-T6 cells. These findings indicated that curcumin might be a potential anti-fibrotic herb for treatment and prevention of hepatic fibrosis through its anti-fibrogenetic and pro-apoptotic effects.

中文摘要…………………………………………………………………… i
中文關鍵字…………………………………………………………………ii
英文摘要……………………………………………………………………iii
英文關鍵字…………………………………………………………………iv

第一章 緒論

前言…………………………………………………………………….…2
1.1 研究背景相關知識
1.1.1 肝臟基本結構、功能與細胞組成……………………………….4
1.1.1.1 基本結構與功能………………………………………… … 4
1.1.1.2 細胞組成……………………………………………………..5
1.1.2 肝纖維化………….………………………………….……... ….. 7
1.1.2.1 肝纖維化的病因……………………………………………..7
1.1.2.2 肝臟星狀細胞在肝纖維化中的作用………………………. 7
1.1.2.3 細胞外基質與肝纖維化……………………………………..9
1.1.2.3.1 細胞外基質……………. ……………………………….9
1.1.2.3.2 細胞外基質的合成與降解…………………………….10
1.1.2.4 TGF-β1與肝纖維化…………………………………………12
1.1.2.5 TGF-β1訊息傳遞路徑………………………………………14
1.1.3 細胞凋亡 (apoptosis)…………………………………… ..........15
1.1.3.1 星狀細胞與細胞凋亡………………………………………16
1.1.3.2 肝細胞凋亡與肝纖維化……………………………………18
1.1.3.3 細胞凋亡機制………………………………………………19
1.1.4 目前西醫對於肝纖維化的治療策略及相關用藥……………...22
1.1.5 中醫對於肝硬化及肝纖維化的認識與治療……………...........23
1.1.5.1 肝硬化中醫古籍記載簡介…………………………………23
1.1.5.2 病因病機……………………………………………………24
1.1.5.3 辨證施治……………………………………………………24
1.1.6 中草藥治療肝硬化、肝纖維化的相關研究…………………...25
1.1.7 薑黃素 (curcumin)及其相關研究簡介………………………...27
1.1.7.1 傳統中醫學方面…………………………………………....27
1.1.7.2 西醫學方面…………………………………………………29
1.1.7.2.1 藥物安全性…………………………………………….29
1.1.7.2.2 生物活性/ 藥理學………………………………….….29
1.2 研究背景與動機….……………………..…………………….….. .32
1.3 研究目的……………………………………………………………34

第二章 材料與方法

2.1 實驗材料
2.1.1 實驗細胞……...……………………………………………..….36
2.1.2 儀器設備………...……………………………………………...36
2.1.3 試劑與藥品……...……………………………………………...38
2.1.4 藥品配製…………...…………………………………………...41
2.2 實驗方法
2.2.1 大鼠肝臟星狀細胞株HSC-T6的培養 (Cell culture)………….45
2.2.2 HSC-T6細胞計數（Cell Counting）…………………………45
2.2.3 HSC-T6細胞存活率分析 (MTT assay)………………………46
2.2.4 HSC-T6細胞膠原蛋白之表現 (Collagen deposition
assay) ...46
2.2.5 HSC-T6細胞α-SMA蛋白質之表現--西方點墨法 (Western
blot)……………………………………………………………47
2.2.6 HSC-T6細胞RNA之萃取…………………………………….48
2.2.7 HSC-T6細胞α-SMA mRNA之表現--反轉錄和聚合酶連鎖
反應 (Reverse transcription-PCR)……………………………49
2.2.8 瓊脂凝膠電泳（Agarose gel electrophoresis）……………….50
2.2.9 細胞凋亡分析--Hoechst 33342 染色法……………………….51
2.2.10 細胞凋亡分析--流式細胞儀測試…………………………….51
2.2.11 細胞色素 c 釋出測定--西方點墨法………………………...52
2.3 統計方法……...…………..………………………………………..53

第三章 結果

3.1 薑黃素 (curcumin)對肝臟星狀細胞HSC-T6活化之研究
3.1.1 薑黃素對TGF-β1誘導星狀細胞膠原蛋白沉積的效應……....55
3.1.2 不同濃度薑黃素對星狀細胞的毒性影響…………………….55
3.1.3 薑黃素對TGF-β1誘導星狀細胞α-SMA蛋白質表現的影響....56
3.1.4 以半定量反轉錄和聚合酶連鎖反應 (semi-quantitative RT-
PCR)分析薑黃素對TGF-β1誘導星狀細胞α-SMA mRNA表現量的影響…………………………………………………...57
3.2 薑黃素對肝臟星狀細胞HSC-T6凋亡之研究
3.2.1 薑黃素對HSC-T6細胞型態的影響…………………………...58
3.2.2 利用Hoechst 33342染色偵測薑黃素對造成HSC-T6細胞凋
亡的影響……………………………………………………... 59
3.2.3 利用流式細胞儀分析薑黃素對HSC-T6細胞週期分佈及造
成其凋亡的影響………………………………………………61
3.2.4 薑黃素對HSC-T6粒線體釋放細胞色素c的影響…………….61

第四章 討論………………………………………………………….......63

4.1 肝纖維化研究的實驗細胞來源--肝臟星狀細胞株HSC-T6……...65
4.2 TGF-β在傷口修復及纖維化疾病中所扮演的角色……..……….68
4.2.1 在傷口修復方面……………………………………………....68
4.2.2 在纖維化疾病方面…………………………………………....70
4.3 TGF-β1對HSC-T6 細胞株的影響…………………………..…...72
4.4 薑黃素對抑制肝臟星狀細胞株HSC-T6 活化之效果……………74
4.5 薑黃素在不同環境下對HSC-T6細胞型態的影響………………..75
4.6 薑黃素對HSC-T6細胞凋亡與細胞週期的影響…………………..76
4.7 不同濃度之薑黃素對抑制肝纖維化之不同效應………………...78
4.8 薑黃素、丹參、川芎與大黃的抗肝纖維化作用………………...78
4.9 未來研究方向……………………………………………………...79

第五章 結論……………...…………………………………………………...81

參考文獻.……………………….….…………………….………………..83

圖表………………………………………………………………………110

圖 目 錄
圖1-1 肝臟損傷時內部結構的變化…………………………………….111
圖1-2 肝臟損傷及復原時肝臟星狀細胞之活化及復原途徑….............112
圖1-3 TGF-β與Smad活化的訊息傳遞路徑………………………….....113
圖1-4 肝細胞凋亡、發炎反應與纖維化間的關係…………………….114
圖1-5 細胞凋亡中由死亡接受器調控的外在路徑…………………….115
圖1-6 細胞凋亡中由粒線體調控的內在路徑…………………..….......116
圖1-7 理想的抗纖維化治療方法……………………………………….117
圖2-1 薑黃 (Curcuma longa Linn.；亞洲地區常稱tumeric或kunyit)藥
材…………………………………………………………………118
圖2-2 丹參 (Salvia miltiorrhiza；Danshen)藥材………………………119
圖3-1 薑黃素對TGF-β1誘發HSC-T6膠原蛋白沉積的效應………..…120
圖3-2 不同濃度薑黃素對HSC-T6細胞存活率的影響………………...121
圖3-3 薑黃素對TGF-β1誘發HSC-T6細胞α-SMA表現之抑制效應…..122
圖3-4 薑黃素對TGF-β1誘發HSC-T6細胞α-SMA mRNA表現之影響..123
圖3-5 薑黃素在不含胎牛血清 (FBS)環境下對HSC-T6細胞型態的影響……………………………....................................................................124
圖3-6 薑黃素在含低量 (2 %)胎牛血清環境下對HSC-T6細胞型態的影響………………………………………………………………………....125
圖3-7 不同濃度之薑黃素對造成HSC-T6細胞凋亡的影響…………...126
圖3-8 薑黃素對HSC-T6細胞週期分佈及造成其凋亡的影響………...129
圖3-9 薑黃素對HSC-T6粒線體釋放細胞色素c的影響…………….....131

表 目 錄
表3-1 薑黃素對HSC-T6細胞週期分佈及造成其凋亡的影響………...132
表4-1 活血化瘀類中藥抗肝纖維化作用比較表………….…………...133

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