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研究生:李庭芳
研究生(外文):Ting-Fang Lee
論文名稱:川芎抑制肝臟星狀細胞株HSC-T6增生之研究
論文名稱(外文):Studies on the antiproliferative activity of Ligusticum chuanxiong in rat hepatic stellate cell line HSC-T6
指導教授:黃怡超黃怡超引用關係林雲蓮林雲蓮引用關係
指導教授(外文):Yi-Tsau HuangYun-Lian Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:傳統醫藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:肝臟星狀細胞肝臟纖維化血小板衍生生長因子川芎細胞凋亡
外文關鍵詞:hepatic stellate cellliver fibrosisPDGFLigusticum chuanxiongapoptosis
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肝臟星狀細胞的增生與活化是造成肝纖維化的重要因素,因此抑制星狀細胞增生並促進星狀細胞凋亡,是目前認為有效抑制肝纖維化的策略之一。血小板生成生長因子-BB (platelet-derived growth factor-BB, PDGF-BB) 是促使星狀細胞增生最有效的裂殖原 (mitogen),本研究欲探討中藥川芎對抑制星狀細胞增生的效果及機轉。
本研究使用肝臟星狀細胞株HSC-T6進行體外 (in vitro)實驗。細胞以PDGF-BB促進增生,並加入川芎抽出物,以BrdU (Bromodeoxy-uridine)偵測川芎對細胞增生的影響,細胞週期分佈的改變則以propidium iodide染色,流式細胞儀測定,細胞週期相關蛋白質cyclin D1,D2,E,A以及p21與p27,使用西方點墨法偵測,JNK磷酸化同樣以西方點墨法測試;Caspase-3活性利用caspase-3基質測定,細胞凋亡使用TUNEL染色,藥物對細胞毒性以MTT測試。
研究結果顯示,PDGF-BB 10 ng/ml可有效促進HSC-T6星狀細胞株增生,達185 ± 6 %,川芎酒精粗抽物在50 μg/ml能顯著地抑制 PDGF-BB造成之細胞增生達53 ± 4 %,其EC50值為45 ± 4 μg/ml。此外,川芎能使 HSC-T6細胞週期在sub G1期及 S期比例增加,CDK (cyclin-dependent kinase)抑制蛋白p21及p27的表現量分別增加432 ± 8 %及 223 ± 7 %。細胞凋亡方面,川芎預處理能造成JNK活化,使caspase-3活性增加197 ± 11 %,並TUNEL染色有陽性反應,顯示造成細胞凋亡。川芎口秦 (tetramethylpyrazine)無法抑制PDGF-BB的作用。川芎粗抽物分劃出的乙酸乙酯可溶物,能夠顯著抑制PDGF-BB造成細胞增生的效果。另外,川芎對初代培養之肝細胞並不造成直接毒性。
川芎對PDGF-BB造成的HSC-T6細胞增生有抑制功效,此抑制效果是藉由細胞週期抑制蛋白p21及p27造成。川芎同時會造成JNK活化與caspase-3活性增加,因而導致HSC-T6細胞凋亡,但並不造成初代培養肝細胞直接毒性本研究顯示川芎有潛力用於治療肝纖維化。
Proliferation and activation of hepatic stellate cells (HSCs) are critical steps in the development of liver fibrosis. Suppression of HSC growth and activation, as well as induction of apoptosis, have been proposed as therapeutic strategies for treatment and prevention of liver fibrosis. Platelet-derived growth factor-BB (PDGF-BB) is the most potent mitogen for the proliferation of HSCs. This study is aimed to elucidate the inhibitory effect of Ligusticum chuanxiong (LC), a Chinese medicinal herb, on PDGF-BB-induced cell proliferation.
This study used the hepatic stellate cell line, HSC-T6 in the in vitro experiments. The cells were stimulated by PDGF-BB in the presence or absence of LC. Proliferation was determined by bromodeoxy uridine incorporation. The cell cycle distribution, affected by PDGF-BB and LC, was determined by using flowcytometric analysis. Cell cycle-related proteins and JNK phosphorylation were evaluated by immunoblotting. Caspase-3 activity was detected by the cleavage activity of cell lysates on caspase-3 substrate. HSC-T6 cell apoptosis was determined by TUNEL staining and the cytotoxic effect of LC on primary hepatocytes was determined by MTT assay.
Results showed that PDGF-BB at 10 ng/ml induced the proliferation of HSC-T6 cells and the proliferation was inhibited by the crude extract of LC at 50 μg/ml by 53 ± 4 %, with the EC50 at 45 ± 4 μg/ml. This effect was accompanied by the change of HSC-T6 cell cycle distribution. The expression of p21 and p27, cell cycle dependent kinase inhibitors, was upregulated. In addition, JNK phosphorylation was increased, caspase-3 activity was enhanced, and apoptosis was observed after treatment with LC at 50 μg/ml. TMP (tetramethylpyrazine), an active compound of LC, was not effective to inhibit the proliferation induced by PDGF-BB. However, ethyl acetate fraction of crude LC significantly inhibited cell proliferation. Moreover, LC did not cause direct cytotoxicity to the isolated rat primary hepatocytes.
This study concludes that LC exerts inhibitory effects of PDGF-BB- induced proliferation on HSC-T6 cells. Expression of p21 and p27 may be the underlying mechanism of this inhibitory effect. LC is able to induce HSC-T6 apoptosis without causing direct cytotxicity to primary hepatocytes. These findings demonstrated that LC might be a potential anti-fibrotic herb for treatment and prevention of hepatic fibrosis.
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行政院衛生署統計室 台灣地區死因統計結果摘要 2002
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