摘要
目的:GCLC基因表达调控有助于在分子水平了解GSH变化的机制,对进一步探索机体抗氧化失衡的机制有重要意义。本实验主要研究GCLC基因上游调控区域(-876^+1)三个相邻E-box元件的作用及探讨E-box元件组合的基因转录作用机制。方法:利用PCR定点缺失法构建多种组合的缺失E-box元件的GCLC上游启动子序列的报告基因载体。将所构建的载体在脂质体介导下瞬时转染大鼠Ⅱ型肺泡上皮细胞(L2细胞),通过比较转染后细胞的荧光素酶活性,分析E-box元件对GCLC基因转录活性的影响。结果:成功构建出多组定点缺失E-box元件的GCLC-Luc基因。在大鼠Ⅱ型肺泡上皮细胞中转染缺失了E-box的GCLC-Luc组较转染GCLC-Luc组均有明显升高(P均<0.05)。结论:三个E-box元件(-804^-779,-729^-724,-590^-585)在GCLC基因的基础状态下的转录表达中都起到一定的抑制作用,可能以转录因子及元件复合物形式抑制GCLC基因的转录调控。此结果揭示GCLC基因上其他E-box元件之间也可能存在着相互作用方式,而非简单的单独作用。
Objectivet Analyses of the expression and regulation of rat GCLC gene can help to understand the mechanisms GSH changes the body＇s resistance at the molecular level which is important to explore the mechanism of oxidative imbalance in the further. This study is to explore the mechanism of gene transcription suppression of E-box element composition by analyzing the three adjacent E-box elements of GCLC （Glutamate-cysteine ligase catalytic subunit） gene in rats. Methods： Using SDM （sire-directed mutagenesis） PCR method constructed a various combinations of E-box elements GCLC promoter sequence upstream of a reporter gene vector. The constructed vectors were transfected to rat lung alveolar type II cell （L2） to determine the relative contribution of E-box on Gclc transcriptional activity. Results： We successfully constructed a multiple fixed missing E-box elements GCLC-Luc genes. In rat type Ⅱ alveolar epithelial cells transfected the missing group than GCLC-Luc group were significantly higher （P〈0.05）. Conclusion： Three E-box elements （-804 - -779, -729 - -724, -590 - -585） in the GCLC＂gene transcription and expression have inhibiting effects to a certain extent and may form complexes to suppress GCLC gene transcription.The results also reveal other E-box elements may interact with each other, rather than simply act alone.
出处
《现代生物医学进展》
CAS
2014年第29期5601-5604,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(30971166)
