地塞米松对人肝癌细胞系SMMC-7721细胞酪氨酸转氨酶活性诱导能力丧失的机制研究

Lost sensibility of tyrosine aminotransferase to dexamethasone in human hepatoma cell line SMMC-7721

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。

Objective：To investigate the mechanism responsible for lost sensibility of tyrosine aminotransferase （TAT） to dexamethasone（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 sequence. TAT mRNA could be induced by Dex,with the maximal induction level being 2. 22-folds in SMMC-7721 cells,which was significantly 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.