摘要
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 in hepatocellular carcinoma cells induced by NF-~B silencing combined with matrine （MT） and the expression of related molecules. Methods The recombinant eukaryotic expression plasmid NF-KB/ P65 siRNA was constructed and transfected into HepG2 cells. RT-PCR was used to detect the efficiency of the constructed RNAi in silencing NF-KB/P65, and the steadily transfected clones of NF-KB/P65 RNAi were selected. The cultured HepG2 cells was randomly divided into four groups： control group, MT group （1.5 g/L）, steadily transfected group and combination group（steadily transfected cells＋MT）. The apoptosis of carcinoma cells was analyzed by flow cytometry; expression of NF-KB/ P65, CD95（Fas）, Smac, and Survivin mRNA and protein in carcinoma ceils was examined by RT PCR and Western blotting analysis. Results The expression of NF-tcB/P65, CD95 （Fas）, Smac, and Survivin mRNA and protein in MT group was significantly increased compared with that in the control group （P〈0.05）. The expression of CD95 and Smac in the steadily transfected group was significantly higher than that in the control group（P〈0. 05）, and the expression of NF-KB/P65 and Survivin was significantly suppressed compared with the control group and the MT group（P〈0.05）. The expression of CD95 and Smac in the combination group was significantly increased compared with that in the other three groups（P〈0.05）, and the expression of NF-KB/P65 and Survivin was significantly lower than that in the MT group（P〈0.05）. The apoptosis rates of the HepG2 cells in the control group, MT group, steadily transfected group, and combination group were 3. 21%, 6.25%0, 11. 82%, and 21.06%, respectively, with significant difference found between different groups（P〈0.05）. Conclusion The apoptosis in hepatocellular carcinoma cells induced by NF-KB RNAi combined with matrine may be related to increased CD95 andSmac expression and decreased NF-KB/P65 and Survivin expression.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2013年第2期148-154,共7页
Academic Journal of Second Military Medical University
