摘要
目的建立化疗药物体外诱导肝癌细胞凋亡的模型,为进一步研究化疗诱导肝癌细胞凋亡的分子机制奠定基础。方法丝裂霉素(MMC)处理人肝癌细胞系HepG2,荧光显微镜和透射电镜观察肝癌细胞的形态学改变,琼脂糖凝胶电泳检测凋亡细胞DNA片段的“梯状”条带,流式细胞仪分析以进一步了解凋亡发生的程度及细胞周期分布情况。结果 MMC能以时间和浓度依赖方式诱导肝癌细胞凋亡,8g·L^(-1)作用24h后肝癌细胞出现凋亡,至48h达高峰。形态学上可见细胞浓缩,核固缩,染色质浓聚聚边等典型凋亡特征;琼脂糖凝胶电泳观察到独特的DNA“梯带”。流式细胞仪检查发现典型的凋亡峰。4g·L^(-1),8g·L^(-1)和16g·L^(-1)药物处理组的细胞凋亡百分率分别为15.83±5.72,20.33±5.73及17.00±6.41,与对照组(1.90±0.48)比较,均有显著性差异(P<0.01);而且8g·L^(-1)组与其他两组比较差别亦有显著性(P<0.05)。空白对照组中,分布在G_1,S,G_2/M各期的细胞比例分别为0.79,0.12,0.09,而经药物处理后相应细胞周期分布为0.53,0.39和0.08,提示肝癌细胞生长明显受阻于G_2/M期。结论 MMC能以时间和浓度依赖方式诱导肝癌细胞凋亡,该凋亡模型的建立将有助于进一步探讨化疗药物诱导肝癌细胞凋亡的分子机制和化疗药物作用机制。
AIM To establish an apoptotic model induced by chemotherapeutic agents in human hepatoma cells in vitro in order to explore the mechanism of chemotherapy-induced apoptosis. METHODS The selected human hepatoma cell line HapG2 was treated with mitomycin C (MMC) at various concentrations and different time. Cells were observed under fiuorescence and electron transmission microscopy. The DNA fragments were also analyzed by agarose gel electrophoresis to testify apoptosis. The flow cytometry (FCM) was used to investigate the apoptotic rate and changes of cell cycle distribution. RESULTS MMC can cause HCC cell line HapG2 to undergo apoptosis in a time and concentration dependent manner. Cell death began approximately at 12 h after administration of 8g·L^(-1) MMC and peaked at 48h following treatment. Under the fluorescence and transmission electron microscopy, the characteristic morphological alteration of apoptosis can be observed such as chromatin margination or condensation, nuclear fragmentation and apoptotic bodies. The typical apoptotic peak was shown by FCM, the apoptotic rates at concentrations of 4g·L^(-1), 8g·L^(-1) and 16g·L^(-1) were 15.83±5.72,20.33±5.73 and 17.00±6.41, respectively which were all significantly higher than that of control group (1.90±0.48). The remarkable difference was also found between the group of 8 g·L^(-1) and other two groups (P<0.05). After administration of chemotherapeutic agents for 24h, the proportions of hepatoma cells distributed in G_1, S, and G_2/M phases of cell cycle were 0.53, 0.39 and 0.08 respectively, while in the non-treated cells, the proportions were 0.79, 0.12 and 0.09 for each corresponding phase. It indicates that the growth of hepatoma cells was arrested in G_2/M phase. CONCLUSION MMC can induce the apoptosis of hepatoma cells in a time and concentration dependent manner.
出处
《世界华人消化杂志》
CAS
2001年第3期268-272,共5页
World Chinese Journal of Digestology
基金
湖南省科委重点资助课题
No.98SSY1008
关键词
丝裂霉素类
药理学
肝肿瘤
肿瘤细胞
脱噬作用
细胞周期
mitomycins/pharmacology
liver neoplasms/pathology
tumor cells, cultured/drug effects
apoptosis/drug effects
cell cycle/drug effects
