【摘要】 目的应用短发夹状RNA介导的RNA干扰研究乙型肝炎病毒X基凶（HBX）和三氧化二砷（As₂O₃）对HepG2细胞p53表达和活性的影响。方法以2μmol/L As₂O₃处理HepG2细胞和表达HBX的HepG2-X,以未处理细胞为对照,提取细胞裂解物或胞核袋解物,采用改进的酶联免疫吸附法检测p53总量和相对活性吸光度。应用脂质体介导具钉HBX序列特异性短发央状RNA表达载体Xi S1、Xi-S2和序列无关对照Xi-S3转染HepG2 X,再次观察As₂O₃处理对p53表达和活性的影响。结果As₂O₀₃作用使HepG2和HepG2 X细胞p53蛋白水平上调和p53相对活性比增强。表达HBX后As₂O₃秀导的p53蛋白水平有所上调,但显著抑制p53相对活性。短发夹状RNA抑制HBX的表达后其对p53的活性抑制得以解除。结论As₂O₃使HepG2细胞p53表达上调和活性增强。应用短发夹状RNA介导的RNA干扰有利于研究HBX的表达对p53表达和活性的影响。HBX表达上调As₂O₃诱导的p53蛋白水平,但显著抑制p53的结合与转录调节活性。更多还原

【Abstract】 Objective To delineate the effects of HBV X gene and of As₂O₃ on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference （RNAi）.Methods HepG2 cells and cells with stable expression of HBV X gene, HepG2-X,were treated with 2μmol/L As₂O₃,and the corresponding untreated cells were used as controls.Cell and nuclear lysates were extracted.Total level and the relative activity absorbance of p53 were detected by modified ELISA.HBV X gene sequence-specific shRNA expression vectors,Xi-S1 and Xi-S2,and sequence-unrelated control Xi-S3 were transfected into HepG2-X.The effect of As₂O₃ on p53 expression and activity were retested.Results Total p53 level was up-regulated and its relative activity ratio was enhanced by As₂O₃ in HepG2 and HepG2-X cells.The total p53 level induced by As₂O₃ was further up- regulated by HBX expression,while its relative activity was significantly suppressed.The suppression was removed after HBX expression was suppressed by shRNA.Conclusion As₂O₃ could up-regulate p53 expression and enhance its activity,shRNA- mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity.HBV X expression could up-regulate p53 gene expression level induced by As₂O₃,while it suppressed the activity of p53.更多还原