苦参碱对人肝癌细胞增殖、细胞周期及细胞凋亡的影响

Effects of matrine on cell proliferation, cell cycle and apoptosis of human hepatocarcinoma cells

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。

AIM： To observe the effects of matrine on cell proliferation, cell cycle phase and apoptosis of human hepatocaroinoma 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）. （1） Effect of matrine on proliferation of human hepatocarcinoma cells： Hepatocarcinoma cells during logarithm growing period were adjusted to 5×10^8 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 methyhhiasolyl tetrasolium assay and enzyme linked immunosorbent assay. There were 6 multiple pores in the negative control group and positive control group. （2）Effect 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 1×10^9 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）.（3）Effect 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, ceils were digested and collected, the concentration was adjusted to 1×10^9 L^-1,then DNA of ceils was extracted according to the instruction on the test kit, 8 g/L agarose gel electrephoresis, observed whether there were DNAladder strap. RESULTS： （1）Matrine can obviously inhibit BEL-7402 ceils 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）.（2）The number of BEL-7402s ceils in G0-G1 phase in the matrine （ 0.5, 1g/L） group were significantly more than that in the contrul group, and in a concentration-depandent manner （ 36.96±1.98,48.41±3.12,27.30±1.11 ;t=-7.37,-11.04,P〈0.01） .③ BEL-7402 ceils showed apoptosis when exposed to 0.5 g/L and t g/L matrine, displaying a distinct apoptotic peak on the histogram. The apoptosis rates were 10.21% and 18.01% respectively. ④ Cell 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 antihepatocarcinoma function by inducing human BEL-7402 cells apoptosis.