二氯乙腈诱导HepG2细胞凋亡及机制研究

目的 研究二氯乙腈(DCAN)对人肝肿瘤HepG2细胞凋亡的作用及其机制。 方法 用二甲基亚砜(DMSO)溶解DCAN,以DMSO处理的HepG2细胞作为对照组, 50和800 μmol/L 的DCAN处理的HepG2细胞作为处理组,采用Cell Counting Kit-8(CCK-8)试剂盒检测细胞活力,流式细胞术(FCM)检测细胞周期分布情况,Annexin V-FITC/PI双标记法测定细胞凋亡率;荧光测定法检测HepG2细胞内天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活性。 结果 DCAN处理浓度和处理时间存在交互效应(P<0.001),与对照组比较,DCAN可明显抑制HepG2增殖,呈现时间和剂量效应(P<0.05);与对照组比较,800 μmol/L DCAN处理组G₀/G₁期比例明显减少,G₂/M期细胞比例明显增加;DCAN诱导的HepG2细胞凋亡随着浓度的增加而增加,呈剂量-效应关系,回归方程为=0.031x+5决定系数(R²)=0.915,F=64.782,P<0.001];DCAN诱导的HepG2细胞内caspase-3酶活性随着处理浓度的增加而增加,呈剂量-效应关系,回归方程为=0.001 6x+1(R²=1.000,F=21 266.064,P<0.001)。 结论 DCAN可抑制HepG2细胞增殖并诱导细胞凋亡,其机制可能与G₂/M期细胞阻滞、抑制RNA和蛋白质的合成并激活细胞内caspase-3凋亡途径有关。

Apoptosis of human hepatoma HepG2 cells induced by dichloroacetonitrile and its mechanism

Objective To study the apoptosis of human hepatoma HepG2 cells induced by dichloroacetonitrile (DCAN) and its related mechanism. Methods Dimethyl sulfoxide (DMSO)-treated HepG2 cells were selected as the control group, while 50 μmol/L and 800 μmol/L DCAN-treated HepG2 cells served as the DCAN-treated groups. The cell proliferation was detected with Cell Counting Kit-8 (CCK-8 ). Cell cycle and apoptosis rate were analyzed by flow cytometry with annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining. The relative activity of caspase-3 in HepG2 cells was examined with fluorospectrophotometer assay. Results The interaction effect existed between DCAN concentration and treatment time (P<0.001). DCAN could significantly inhibit the proliferation of HepG2 cells in a dose- and time-dependent manner compared with the control group (P<0.05). The proportion of HepG2 cells in G₀/G₁ phase was decreased in the 800 μmol/L DCAN-treated group compared with the control group, while the proportion of HepG2 cells in G₂/M phase was increased. Under the DCAN concentrations, the apoptosis rate of HepG2 cells were increased in a dose-dependent manner, with the regressionequation =0.031x+5 (R²=0.915, F=64.782, P<0.001), while the caspase 3 activity was also increased in a dose-dependent manner, with regression equation =0.0016x+1 (R²=1.000,F=21,266.064,P<0.001). Conclusions DCAN can inhibit the proliferation of HepG2 cells and induce their apoptosis, and the mechanism may be related to the arrest of G₂/M phase, inhibition of RNA and protein synthesis and activation of caspase-3 pathway.