芬太尼及Ly294002对人肝癌HepG2细胞增殖及迁移的抑制作用及其机制

摘要

Objective To explore the inhibitory effects of fentanyl and Ly294002 on the proliferation and migration of human hepatoma HepG2 cells and its mechanism.Methods HepG2 cells at logarithmic growth phase were divided into fentanyl group,Ly294002 group,combination group and control group.The cells of the fentanyl group,Ly294002 group,combination group and control group were treated by 500 ng/ml fentanyl,25 μmol/L Ly294002,fentanyl(500 ng/ml) combined with Ly294002(25 μmol/L),and medium with same volume respectively.Then methyl thiazolyl tetrazolium assay was used to evaluate the proliferation activity,characterized by absorbency (A value),wound healing assay to detect the migration activity,then the recovery rate was calculated,flow cytometer to observe the cell cycle changes and cell apoptosis,and realtime quantitative PCR to determine the expression levels of phosphate and tension homology deleted on chromsome ten(PTEN) mRNA and nuclear factor kappa B(NF-κB) mRNA.Results In the fentanyl group,Ly294002 group and combination group,the A values,recovery rates,ratios of cells at(S+G2/M) phase,and relative expression levels of NF-κB mRNA were lower than those in the control group(all P<0.05),and the ratios of cells at G0/G1phase,apoptosis rates,and relative expression levels of PTEN mRNA were higher than those in the control group(all P<0.05).The A value,recovery rate,ratio of cells at(S+G2/M) phase,and relative expression of NF-κB mRNA were lower,and the ratio of cells at G0/G1phase,apoptosis rate,and relative expression level of PTEN mRNA were higher in the combination group than those in the fentanyl group and Ly294002 group(P<0.05).Conclusion Either Fentanyl or Ly294002 can inhibit the proliferation and migration of human hepatoma HepG2 cells,achieving synergistic effects, which may be related to protein kinase B(Akt) signal transduction pathway.%目的 探讨芬太尼及Ly294002对人肝癌HepG2细胞增殖、迁移的抑制作用及其机制.方法 将对数生长期的人肝癌HepG2细胞分为芬太尼组、Ly294002组、联合组和对照组,分别给予500 ng/ml 芬太尼、25 μmol/L Ly294002、500 ng/ml芬太尼联合25 μmol/L Ly294002、等量培养基进行干预.用四甲基偶氮唑蓝法检测细胞增殖活性,用吸光度A值表示,划痕实验观察细胞迁移能力并计算愈合率,流式细胞仪观察细胞周期和细胞凋亡情况,实时荧光定量PCR法检测细胞中人第10号染色体缺失的磷酸酶(PTEN)mRNA 和核转录因子 κB(NF-κB)mRNA 的表达水平.结果 芬太尼组、Ly294002组和联合组的A值、愈合率、(S+G2/M)期细胞比例、NF-κB mRNA相对表达水平均低于对照组(均P<0.05),而G0/G1期细胞比例、凋亡率、PTEN mRNA相对表达水平高于对照组(P<0.05);联合组的A值、愈合率、(S+G2/M)期比例、NF-κB mRNA相对表达水平低于芬太尼组及Ly294002组(P<0.05),而G0/G1期细胞比例、凋亡率、PTEN mRNA相对表达水平高于芬太尼组及Ly294002组(P<0.05).结论 芬太尼和Ly294002均可抑制人肝癌HepG2细胞增殖和迁移,且两者有协同作用,这可能均与蛋白激酶B(Akt)信号转导通路有关.