【摘要】 目的:观察传统中药苦参的主要成分苦参碱对人肝癌细胞体外增殖、细胞周期及细胞凋亡作用的影响,探讨其对肝癌可能的作用机制。方法:实验于2004-05/2004-11在锦州医学院免疫与病原生物学教研室完成。选用人肝癌细胞株(BEL-7402),分设苦参碱0.5,0.75,1g/L浓度组,阴性对照组和阳性对照组(顺铂组)。①苦参碱对人肝癌细胞增殖作用的影响:将对数生长期的人肝癌细胞调至5×108L-1,按每孔100μL接种到96孔板内,培养24h后加入不同浓度(0.5,0.75,1g/L)的药物,继续培养72h。采用四甲基偶氮唑盐法最后用酶标仪测A值。实验同时设阴性对照组和阳性对照组(顺铂组),每组设6复孔。②苦参碱对人肝癌细胞周期的影响:将对数生长期的细胞用苦参碱(0.5,1g/L)处理48h后,消化并收集细胞,冲液后固定,4℃过夜。次日再冲洗2次,染色,调整细胞浓度至1×109L-1,流式细胞仪检测,按FACSort软件多正态拟合程序进行曲线拟合分析,计算DNA含量,得出细胞周期(G1、S、G2+M)的百分比。③苦参碱对人肝癌细胞DNALadder条带出现的影响:将对数生长期的细胞用苦参碱(0.5g/L、1g/L)处理48h后,消化并收集细胞,调整细胞浓度至1×109L-1,然后按细胞DNA提取试剂盒说明操作,提取细胞DNA,8g/L琼脂糖凝胶电泳,观察有无DNAladder条带出现。结果:①苦参碱在0.5,0.75,1g/L浓度时均能明显抑制人肝癌细胞增殖,抑制率分别为17.24%、26.05%和50.77%,存在着明显地浓度依赖关系,与对照组相比差异显著(P<0.01)。②苦参碱0.5,1g/L浓度组G0～G1期细胞数量明显高于对照组,并呈浓度依赖关系(36.96±1.98,48.41±3.12,27.30±1.11;t=-7.37,-11.04,P<0.01)。③苦参碱(0.5,1g/L)作用后可使人肝癌细胞发生凋亡,直方图上显示典型的细胞凋亡峰,凋亡率分别为10.21%、18.01%。④提取细胞DNA、电泳,可见苦参碱1g/L组出现典型的梯形条带,苦参碱0.5g/L组梯形条带不明显,而对照组未见梯型条带。结论:苦参碱对人肝癌细胞有明显地抑制作用,并存在着明显地浓度依赖关系;苦参碱可能是通过将人肝癌细胞周期阻滞在G0～G1期,抑制其增殖,通过诱导人肝癌细胞发生凋亡等,发挥其抗肝癌作用。更多还原

【Abstract】 AIM: To observe the effects of matrine on cell proliferation, cell cycle phase and apoptosis of human hepatocarcinoma cells and probe into its anti-cancer mechanisms.METHODS: The experiment was conducted in the Staff Room of Immunology and Pathobiology, Jinzhou Medical College from May to November 2004. Human cell strain BEL-7402 were divided into matrine 0.5, 0.75, 1 g/L group, negative control group and positive control group (cisplatin group).①Effect of matrine on proliferation of human hepatocarcinoma cells: Hepatocarcinoma cells during logarithm growing period were adjusted to 5×108 L-1, inoculated to 96 pore plates with 100 μL in each pore, drugs of different concentration were added 24 hours after cultivating, then cultivated for another 72 hours. A value was measured with methylthiazolyl tetrazolium assay and enzyme linked immunosorbent assay. There were 6 multiple pores in the negative control group and positive control group.②Effect of matrine on cell cycle of human hepatocarcinoma: Hepatocarcinoma cells during logarithm growing period were done with matrine (0.5,1 g/L), 48 hours after that, cells were digested and collected, and fixed after flushed with fluid, then lasted for a night at 4 ℃ and flushed twice in the next day, then stained. The concentration of cells was adjusted to 1×109 L-1, detected with flow cytometer, DNA content was caculated through curve fitting analysis according to the FACSort multiple normality fitting program, so as to obtain the persentage of cell cycle(G1?S?G2+M).③Effect of matrine on the strap of human hepatocardinoma DNAladder: Hepatocarcinoma cells during logarithm growing period were done with matrine(0.5,1 g/L), 48 hours after that, cells were digested and collected, the concentration was adjusted to 1×109 L-1,then DNA of cells was extracted according to the instruction on the test kit, 8 g/L agarose gel electrophoresis, observed whether there were DNAladder strap. RESULTS: ①Matrine can obviously inhibit BEL-7402 cells propagation at the concentration of 0.5, 0.75 and 1 g/L, and the inhibiting rates were 17.24%, 26.05% and 50.77% respectively, which was in a obvious concentration-dependent manner, and there were significant differences in comparison with the control group (P < 0.01).②The number of BEL-7402s cells in G0-G1 phase in the matrine ( 0.5, 1g/L) group were significantly more than that in the control group, and in a concentration-dependent manner(36.96±1.98,48.41±3.12,27.30±1.11;t=-7.37,-11.04,P < 0.01).③BEL-7402 cells showed apoptosis when exposed to 0.5 g/L and 1 g/L matrine, displaying a distinct apoptotic peak on the histogram. The apoptosis rates were 10.21% and 18.01% respectively.④Cell DNA extraction was performed by eletrophoresis, and typical DNA ladder strap was found in the matrine (1 g/L) group, while no distinct DNA ladder was found in the 0.5 g/L group and the control group.CONCLUSION: Matrine has a significant inhibiting effect on human hepatocarcinoma cells in a obvious concentration-dependent manner; Matrine inhibits the proliferation of the cells by blocking the hepatocarcinoma cell cycle to G0-G1 phase, and develops its anti-hepatocarcinoma function by inducing human BEL-7402 cells apoptosis.更多还原