古抑菌素A对人肝癌细胞株HepG2增殖和凋亡的影响及其机制研究

Effect of Histone Deacetylase Inhibitor TSA on Proliferation and Apoptosis of Human Hepatocellular Carcinoma Cell Line HepG2 and the Study of Its Mechanism

目的探讨去乙酰化转移酶抑制剂古抑菌素A(TSA)对人肝癌细胞株HepG2增殖及凋亡的影响。方法体外培养人肝癌细胞株HepG2,以不同浓度的TSA(5、50、250、500、1000、2000nmol/L)处理24h后在倒置显微镜和透射电镜下观察HepG2形态的变化,用CCK-8法检测细胞增殖活性。用TUNEL法检测对照组(加等量DM-SO)和500nmol/LTSA组的细胞凋亡率,用免疫细胞化学法检测两组增殖凋亡调控相关基因caspase-3、bax和bcl-2的蛋白表达。结果倒置显微镜下,经TSA处理的细胞增殖速度显著减慢;透射电镜下,HepG2出现凋亡早期改变。CCK-8法测定结果显示,对照组和不同浓度TSA(5、50、250、500、1000、2000nmol/L)组HepG2的增殖活性分别为(0.637±0.032)、(0.552±0.016)、(0.499±0.031)、(0.393±0.007)、(0.321±0.026)、(0.277±0.010)和(0.275±0.015),组间比较差异有统计学意义(P<0.01);且不同浓度的TSA对HepG2的增殖抑制作用有明显的剂量依赖关系。500nmol/LTSA组细胞凋亡率显著高于对照组,且其凋亡相关蛋白caspase-3和bax的表达较对照组显著增强,差异有统计学意义(P<0.05);而两组bcl-2的蛋白表达间差异无统计学意义(P>0.05)。结论 TSA可通过增加caspase-3、bax的蛋白表达,诱导细胞凋亡而对人肝癌细胞株HepG2具有增殖抑制作用。

Objective To investigate the effect of TSA,an inhibitor specific for histone deacetylase,on the proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2.Methods The cultured human hepatocellular carcinoma cells HepG2 were treated with different concentrations of TSA（5,50,250,500,1 000,2 000 nmol/L）for 24 hours.Then cell morphology was observed by the inverted light microscope and electron-microscopy,cell proliferation was detected using CCK-8 approach.Cell apoptosis rate of the control group （treated by the same amount of DMSO） and the group treated by 500 nmol/L TSA was determined using TUNEL approach.The expression of proliferation and apoptosis associated proteins of caspase-3,bax and bcl-2 were determined by immunocytochemistry approach.Results After treated with TSA,the proliferation of HepG2 cells reduced significantly,and HepG2 cells showed early apoptosis under electron-microscope.The proliferation activity of HepG2 cell in control group and different concentrations of TSA （5,50,250,500,1 000,2 000 nmol/L） group were （0.637±0.032）,（0.552±0.016）,（0.499±0.031）,（0.393±0.007）,（0.321±0.026）,（0.277±0.010）,（0.275±0.015）,respectively,and the difference was significant（P0.01）.Different concentrations of TSA inhibited the proliferation of HepG2 differently.After treated with 500 nmol/L TSA,apoptosis rate and the expressions of apoptosis-related protein caspase-3 and bax were significantly increased compared with the control group,but the level of bcl-2 did not change significantly （P0.05）.Conclusion TSA can inhibit the proliferation of the hepatocarcinoma HepG2 cells through inducing cell apoptosis and increasing the expression of protein caspase-3 and bax.