不同浓度腐胺对人肝细胞增殖及凋亡的影响

Effects of different concentrations of putrescine on proliferation and apoptosis in human hepatocyte cell

目的探讨不同浓度腐胺对体外培养人正常肝细胞增殖、凋亡的影响。方法将体外培养的人正常肝细胞株LO2分为腐胺组与对照组,不同浓度腐胺组以含腐胺浓度分别为0.25、0.50、1.00、10.00、20.00、40.00、80.00、160.00、320.00、640.00μg/mL完全培养基培养细胞,对照组以不添加腐胺完全培养基培养细胞。对照组与不同浓度腐胺组处理的LO2细胞培养12 h后,再以四唑化合物电子耦联显色法（MTS）、流式细胞技术（FCM）分别测定细胞的增殖活性（用吸光度值表示）与凋亡率,并对两组数据进行相关性分析。结果 MTS结果显示,0.25、0.50、1.00、10.00、20.00μg/mL浓度腐胺组LO2细胞吸光度值均较对照组（0.474±0.022）升高,差异有统计学意义（P〈0.05）,并且1.00μg/mL浓度时达到峰值（0.834±0.012）;80.00μg/mL及以上浓度腐胺组LO2细胞吸光度值较对照组降低,差异有统计学意义（P均〈0.01）;40.00μg/mL腐胺组LO2细胞吸光度值（0.477±0.009）与对照组比较,差异无统计学意义（P〉0.05）。流式细胞技术结果显示,0.25、0.50、1.00、10.00、20.00μg/mL浓度腐胺组LO2细胞凋亡率均较对照组（15.23±1.82）%降低,差异有统计学意义（P〈0.05）,并且1.00μg/mL腐胺组细胞凋亡率达到最低值（2.30±1.00）%;80.00μg/mL及以上浓度腐胺组LO2细胞凋亡率较对照组升高,差异有统计学意义（P均〈0.01）;40.00μg/mL腐胺组LO2细胞凋亡率（16.10±1.45）%与对照组比较,差异无统计学意义（P〉0.05）。相关性分析结果显示,各组浓度腐胺处理LO2细胞其细胞增殖与凋亡率呈负线性相关关系（r=-0.989,P=0.000）。结论低浓度（0.25-20.00μg/mL）腐胺对人正常肝LO2细胞增殖有促进作用,而较高浓度（80.00μg/mL及以上）的腐胺则出现明显的诱导凋亡作用,低浓度腐胺促进细胞增殖可能是通过抑制细胞凋亡而实现。

Objective To explore the effects of different concentrations of putrescine on proliferation and apoptosis in human hepatocyte cell in vitro. Methods Human hepatocyte cell（ LO2） cultured in vitro were randomly divided into putrescine groups and control group,and cells in putrescine groups respectively were cultured by 0. 25,0. 50,1. 00,10. 00,20. 00,40. 00,80. 00,160. 00,320. 00,640. 00 μg /mL putrescine for 12 h,relatively cells in control group were without putrescine. The absorbance values were measured by MTS assay,the apoptosis rates assay were performed by Annexin V /PI in flow cytometry.Correlation analysis was done with the relation of absorbance values and apoptosis rate. Results MTS assay showed that the absorbance values of LO2 in 0. 25,0. 50,1. 00,10. 00,20. 00 μg /mL putrescine groups were higher than that of control group（ 0. 474 ± 0. 022）,there were statistically significant differences（ P〈0. 05）,and that of 1. 00 μg /mL reached the peak（ 0. 834 ± 0. 012）. The absorbance values in 80. 00 μg /mL and higher concentration putrescine groups were lower than that of control group,there were statistically significant differences（ P〈0. 01）. The difference in absorbance value between 40. 00 μg /mL（ 0. 477 ±0. 009） and the control group was no statistically significant difference（ P〈0. 05）. Flow cytometry assay showed that the apoptosis rates of LO2 cell treated with concentrations of 0. 25,0. 50,1. 00,10. 00,20. 00 μg /mL putrescine were lower than that of control group（ 15. 23 ± 1. 82） %,there were statistically significant differences（ P〈0. 05）. The apoptosis rate of 1. 00 μg /mL putrescine group reached the minimum value（ 2. 30 ± 1. 00） %. The apoptosis rates in 80. 00 μg /mL and higher concentrations putrescine groups were greater than that of control group. There were statistically significant differences（ P〈0. 05）. The difference in apoptosis rates between 40. 00 μg /mL（ 16. 10 ± 1. 45） % and the control group was no statistically significant（ P〉0. 05）. There was negative correlation between proliferation and apoptosis rate in LO2 cell exposed in the same concentrations of putrescine（ r =- 0. 989,P = 0. 000）. Conclusions The low concentrations of putrescine（ 0. 25-20. 00 μg /mL） can promote the growth of human hepatocyte cell.However,the high concentrations of putrescine（ 80. 00μg /mL and more） can induce LO2 cell apoptosis.The effect of cell proliferation possibly can be attributed to blocking apoptosis.