- 學位論文
硫辛酸誘導活化肝星狀細胞凋亡之研究
Induction of activated hepatic stellate cell apoptosis by α-lipoic acid
摘要
摘要 肝臟是人體代謝各種物質的重要器官，當肝臟慢性發炎，肝星狀細胞會被活化造成肝臟的纖維化甚至肝硬化，這樣的過程常發生在慢性肝炎的患者身上。若能以誘導肝星狀細胞進行細胞凋亡，則能以不傷害人體、不引起發炎的方式治療病人。硫辛酸為一種天然的抗氧化劑，本研究以硫辛酸加入培養液中，培養體外的活化肝星狀細胞，觀察硫辛酸是否會誘導活化肝星狀細胞的凋亡。實驗以代表正常肝細胞的Clone 9當作細胞毒性控制組，以MTT assay觀察細胞存活率的變化、西方墨點法分析細胞凋亡分子caspase 9的表現情形、細胞週期分析是否有細胞週期停滯的情形、ROS與粒腺體膜電位分析測試粒腺體是否有損傷。實驗結果顯示，在1 mM硫辛酸下，活化肝星狀細胞的存活率於24小時後會明顯的降低，卻不會對clone9造成影響。於250 ?嵱的硫辛酸作用下，HSC的caspase 9表現明顯的高過clone 9。細胞週期分析顯示HSC有很明顯的G0/G1期停滯，然而HSC和clone 9在硫辛酸的作用下並沒有大量的產生 ROS，反而有減少的趨勢，且細胞膜電位皆無下降的趨勢。因此證實硫辛酸誘導肝星狀細胞凋亡係經由細胞週期停滯於G0/G1且經由內生性途徑的方式造成；或許因硫辛酸自身的抗氧化特性，故其過程中不會導致粒腺體膜電位下降及超氧陰離子(O2 -)、過氧化氫(H2O2 )及羥基自由基(˙OH)相關的ROS上升。
並列摘要
Abstract Liver plays an important role in the metabolism and detoxification. Inflammation and chronic liver diseases lead to the activation of hepatic stellate cells (HSCs), resulting in liver fibrosis and cirrhosis. Therefore one therapy strategy for chronic liver diseases is to induce apoptosis of HSCs so that patients would not suffer from inflammatory reactions. α-Lipoic acid is a natural antioxidant. In this study, the apoptotic effect of α-lipoic acid on HSCs in vitro was investigated by MTT assay, western blot, cell cycle analysis, and detection of ROS and mitochondrial membrane potential. The results indicated that the viability of HSCs was decreased followed by the treatment of 1mM or more α-lipoic acid for 24 hours. The caspase-9 protein level of HSCs treated with 250 ?嵱 α-lipoic acid was significantly higher than that of Clone9 (toxicity control). The cell cycle analysis demonstrated α-lipoic acid induced G0/G1-phase arrest on HSC. However, the intracellular ROS was not significantly increased but decreased on both HSC and Clone9, and the mitochondrial membrane potential was not dropped but risen on both HSC and Clone9. The data revealed that α-lipoic acid resulted in the G0/G1 cell cycle arrest and induced the apoptosis of HSC by the intrinsic pathway. Perhaps the antioxidative capacity of α-lipoic acid contributed to the high mitochondrial membrane potential and the low ROS level.