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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
李忆东,刘宇健,卢建*
0
(第二军医大学基础部病理生理学教研室,上海 200433)
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。
关键词:  糖皮质激素  受体  酪氨酸转氨酶  转录激活
DOI:10.3724/SP.J.1008.2008.00630
投稿时间:2008-01-29修订日期:2008-03-03
基金项目:国家自然科学基金重点项目(39730210).
Lost sensibility of tyrosine aminotransferase to dexamethasone in human hepatoma cell line SMMC-7721
LI Yi-dong,LIU Yu-jian,LU Jian*
(Department of Pathophysiology,College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433,China)
Abstract:
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.
Key words:  glucocorticoids  receptor  tyrosine aminotransferase  transcriptional activation