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机构地区:[1]第二军医大学基础部病理生理学教研室,上海200433
出 处:《第二军医大学学报》2008年第6期630-633,共4页Academic Journal of Second Military Medical University
基 金:国家自然科学基金重点项目(39730210).~~
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。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.
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