【摘要】 目的:通过测定人肝癌SMMC 7721细胞酪氨酸转氨酶(TAT)cDNA序列,并观察其糖皮质激素受体(GR)通路是否存在异常,以探讨地塞米松(Dex)丧失诱导SMMC-7721细胞TAT活性的机制。方法:RT-PCR扩增SMMC-7721细胞全长TAT cDNA并进行测序;采用实时定量PCR观察Dex对SMMC-7721细胞TAT mRNA表达的影响,并与HepG2细胞作对照;采用电穿孔法将GR特异性报告基因GRE-tk-LUC及GRE-MMTV CAT分别瞬时转入SMMC-7721细胞中,观察Dex对荧光素酶(LUC)和氯酶素乙酰转移酶(CAT)表达的影响,并与HepG2细胞作对照。结果:SMMC-7721细胞TAT cDNA中第576位碱基存在同义突变;Dex能够诱导SMMC-7721细胞中TAT mRNA的表达,最大诱导倍数为2.22,明显低于HepG2细胞(15.1倍,P<0.01);Dex诱导了SMMC-7721细胞LUC及CAT的表达,与HepG2细胞无显著差异。结论:Dex对SMMC-7721细胞TAT mRNA诱导水平降低,可能是TAT活性不增高的主要原因。更多还原

【Abstract】 Objective:To investigate the mechanism responsible for lost sensibility of tyrosine aminotransferase(TAT)to dexam- ethasone(Dex)in human hepatoma cell line SMMC-7721 through examining the cDNA sequence of TAT and the status of glucocorticoid receptor(GR)pathway.Methods:The TAT cDNA fragment containing the full length of coding sequence was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and was sequenced.The expression of TAT mRNA was determined by real-time quantitative PCR to observe the influence of Dex on expression of TAT mRNA in SMMC-7721 cells.The experiement with HepG2 cells was performed as the control.Reporter genes(GRE-tk-LUC and GRE-MMTV-CAT)were transiently transfected into SMMC-7721 cells by electroporation.The induction efficiencies of LUC and CAT genes expression by Dex were examined and compared between SMMC-7721 cells and HepG2 cells.Results:The results showed that there was a same-sense mutation(Gln576Gln)in TAT cDNA se- quence.TAT mRNA could be induced by Dex,with the maximal induction level being 2.22-folds in SMMC-7721 cells,which was signifi- cantly lower than that in HepG2 cells(15.1-fold increase,P<0.01).Dex induced the expression of LUC and CAT genes in SMMC- 7721 cells as well as the HepG2 cells.Conclusion:The induction efficiency of Dex for expression of TAT mRNA is decreased in SMMC- 7721 cells,which might be due to the unchanged activity of TAT.更多还原