摘要
AIM： Thioacetamide （TAA） has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS： The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA- induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA- induced clone 9 cells were measured by Western blot. RESULTS： The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died （MTT assay） within 24 h after 100 rag/1 TAA was applied. Apoptotic cell percentage （TUNE1 assay） and caspase 3 activities were highest after 100 rag/1 TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. CONCLUSION： These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.
瞄准：Thioacetamide (TAA ) 在然而，学习肝纤维变性和肝硬化被使用了在肝的导致 TAA 的 apoptosis 的机制仍然是不清楚的。肝的上皮的房间线克隆 9 与 TAA 有教养、对待调查细胞死亡的原因。方法：导致 TAA 的克隆的房间生存能力 9 个房间用 MTT 被决定试金。在导致 TAA 的克隆的全部的细胞的 GSH 9 个房间用 Tietze 试金的细微修正被测量。caspase 的活动 3 在导致 TAA 的克隆， 9 个房间被 DEVD-p-nitroanaline 的劈开监视。TUNEL 试金和流动血细胞计数被申请 DNA 破碎的决心和在导致 TAA 的克隆的 apoptosis 的比例 9 个房间分别地。caspase 的改变 3，坏，在导致 TAA 的克隆的 Bax 和 Phospho-P53 内容 9 个房间被西方的污点测量。结果：试验性的数据显示 TAA 在剂量引起了老鼠肝的上皮的房间线克隆 9 细胞死亡 -- 并且时间依赖者举止；60% 房间死了(MTT 试金) 在在 100 mg/L 以后的 24 h 以内， TAA 被使用。Apoptotic 房间百分比(TUNEL 试金) 和 caspase 在 100 mg/L TAA 为 8 h 被增加以后， 3 项活动最高。在在 TAA 处理以后的 caspase 内容的 GSH 和举起的版本经由氧化应力和 caspase 依赖的机制导致了克隆 9 房间 apoptosis。在克隆 9 房间的 phospho-p53， Bax 和坏蛋白质表情在 TAA 处理以后被增加。结论：这些结果表明 TAA 激活 p53，增加 caspase 3， Bax 和坏蛋白质内容，也许从线粒体和膜的崩溃引起细胞色素 c 的版本，导致房间的 apoptosis。
基金
Supported by the National Science Council, Taiwan, No.92-2317B-259-001
