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目的探讨NF-κB沉默联合苦参碱(matrine,MT)诱导人肝癌细胞HepG2凋亡及相关分子的表达。方法构建NF-κB/P65 siRNA真核表达载体转染人肝癌细胞HepG2,RT-PCR检测NF-κB/P65基因沉默效果,并筛选出NF-κB/P65基因沉默稳定转染细胞株。将实验分为对照组、苦参碱组(1.5 g/L)、稳转细胞组和联合组(稳转细胞+苦参碱),流式细胞仪检测细胞凋亡率;分别用RT-PCR法和蛋白质印迹法检测细胞NF-κB/P65、CD95(Fas)、Smac、Survivin的mRNA和蛋白水平表达。结果苦参碱组NF-κB/P65、CD95、Smac和Survivin的表达上调,与对照组比较差异具有统计学意义(P<0.05);稳转细胞组CD95和Smac的表达上调,与对照组比较差异有统计学意义(P<0.05),NF-κB/P65和Survivin的表达下调,与对照组、苦参碱组比较差异有统计学意义(P<0.05);联合组CD95和Smac的表达上调,与对照组、苦参碱组、稳转细胞组比较差异有统计学意义(P<0.05),NF-κB/P65和Survivin的表达下调,与苦参碱组比较差异有统计学意义(P<0.05)。各组细胞凋亡率分别为3.21%、6.25%、11.82%、21.06%,组间差异均有统计学意义(P<0.05)。结论 NF-κB沉默联合苦参碱诱导人肝癌细胞HepG2凋亡可能与CD95和Smac的表达上调及NF-κB/P65和Survivin的表达下调有关。
Objective To investigate the apoptosis of HepG2 cells induced by NF-κB combined with matrine (MT) and its related molecules. Methods The eukaryotic expression vector of NF-κB / P65 siRNA was constructed and transfected into HepG2 cells. The silencing effect of NF-κB / P65 gene was detected by RT-PCR and the cell lines stably transfected with NF-κB / P65 gene were screened out. The experiment was divided into the control group, the matrine group (1.5 g / L), the stable cell group and the combined group (steady-state cell + matrine), and the apoptosis rate was detected by flow cytometry; The mRNA and protein expression of NF-κB / P65, CD95 (Fas), Smac and Survivin were detected by Western blotting and Western blot. Results The expression of NF-κB / P65, CD95, Smac and Survivin were up-regulated in matrine group, which was significantly different from the control group (P <0.05). The expression of CD95 and Smac in metastatic cells was up-regulated The difference was statistically significant (P <0.05), the expression of NF-κB / P65 and Survivin were down-regulated compared with the control group and matrine group (P <0.05); the expression of CD95 and Smac in the combined group was increased (P <0.05). The expression of NF-κB / P65 and Survivin were down-regulated in the matrine group compared with the matrine group (P <0.05), and the difference was statistically significant <0.05). The apoptosis rate of each group was 3.21%, 6.25%, 11.82% and 21.06%, respectively. The differences among the groups were statistically significant (P <0.05). Conclusions The combination of NF-κB silencing and matrine-induced HepG2 apoptosis may be related to the upregulation of CD95 and Smac and the down-regulation of NF-κB / P65 and Survivin expression.