论文部分内容阅读
目的了解丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)对p53抑制甲胎蛋白(AFP)基因表达的影响及分子机制。方法采用质粒转染技术及微粒体酶免疫法观察p53蛋白对Huh7肝癌细胞AFP表达的抑制作用及HCV NS5A对其抑制作用的影响;蛋白印迹实验观察HCV NS5A对p53蛋白表达的影响;谷胱甘肽转移酶沉淀实验鉴定HCV NS5A与p53蛋白能否相互作用形成复合物。结果转染pRc/CMV空质粒的Huh7细胞上清液AFP浓度为(14 322±2412)ng/ml,转染pCNS5A质粒的Huh7细胞上清液的AFP 浓度为(13 843±3218)ng/ml,两组问t=1.42,P>0.05;转染pC53-NS3质粒的Huh7细胞上清液的AFP 浓度为(10 241±1326)ng/ml,与上述两组比较,t值分别为2.41及2.38,P值均<0.05;pCNS5A和pC53- NS3共转染者AFP浓度为(14 582±1238)ng/ml,与pC53-NS3单独转染组比较,t=3.12,P<0.01; pCNS5A和pC53-NS3共转染者和pC53-NS3单独转染者Huh7细胞p53蛋白表达无变化。在谷胱甘肽转移酶沉淀实验中,加入谷胱甘肽-p53融合蛋白后出现HCV NS5A蛋白条带,而仅加入谷光甘肽者则未出现此条带。结论p53蛋白能抑制Huh7细胞AFP的表达,HCV NS5A能减轻p53蛋白对AFP表达的抑制作用。HCV NS5A不影响p53蛋白的表达但能与p53蛋白结合形成复合物是使p53功能失活的分子机制。
Objective To investigate the effect of hepatitis C virus nonstructural protein 5A (NS5A) on the expression of p53 -inhibiting alpha-fetoprotein (AFP) gene and its molecular mechanism. Methods The effect of p53 protein on the expression of AFP in Huh7 hepatocarcinoma cells and the inhibition of HCV NS5A were observed by plasmid transfection and microsomal enzyme immunohistochemistry. The effect of HCV NS5A on p53 protein expression was observed by Western blotting. Peptide transferase pelleting experiments identified whether HCV NS5A interacts with the p53 protein to form a complex. Results The concentration of AFP in supernatant of Huh7 cells transfected with pRc / CMV empty plasmid was (14 322 ± 2412) ng / ml and that of Huh7 supernatant transfected with pCNS5A plasmid was (13 843 ± 3218) ng / ml , T = 1.42, P> 0.05. The AFP concentration of Huh7 cell supernatant transfected with pC53-NS3 plasmid was (10 241 ± 1326) ng / ml, compared with the two groups, the t values were 2.41 and 2.38, P <0.05). The concentration of AFP in pCNS5A and pC53-NS3 co-transfection group was (14 582 ± 1238) ng / ml, which was significantly lower than that in pC53-NS3 transfection group There was no change in p53 protein expression in Huh7 cells transfected with pC53-NS3 and pC53-NS3 alone. In glutathione transferase precipitation experiments, the addition of glutathione-p53 fusion protein showed a band of the HCV NS5A protein, but only the addition of glutathione did not show this band. Conclusion p53 protein can inhibit AFP expression in Huh7 cells. HCV NS5A can reduce the inhibitory effect of p53 protein on AFP expression. HCV NS5A does not affect the expression of p53 protein but binds to p53 protein to form a complex, which is the molecular mechanism of p53 function inactivation.