腺病毒E1A蛋白对大鼠肺泡上皮细胞谷胱甘肽活性的影响及其机制探讨

The influence of adenovirus latent infection on glutathione and antioxidative enzymes in rat alveolar epithelial cells

目的研究腺病毒E1A蛋白潜伏感染对大鼠肺泡上皮细胞谷胱甘肽（GSH）活性的影响及其机制。方法构建稳定表达腺病毒E1A蛋白的大鼠肺泡上皮细胞，观察氧化剂刺激时细胞内GSH含量变化，检测GSH合成限速酶γ-谷氨酰半胱氨酸合成酶（γ-GCS）催化活性的变化，通过观察γ-GCS催化亚单位（GCLC）基因蛋白表达水平、mRNA表达水平和5’-上游调控序列调控的荧光素酶活性的改变，了解影响酶活性改变的机制。结果腺病毒E1A蛋白表达抑制氧化应激时GSH的合成，抑制γ-GCS催化活性和蛋白表达水平，抑制GCLC基因mRNA表达，与5’-上游调控序列报导系统获得的结果一致。结论腺病毒潜伏感染可能通过抑制GCLC基因5’-上游调控序列的促转录活性，抑制了γ-GCS的表达和催化活性，从而抑制了氧化应激时GSH的合成，削弱机体的抗氧化作用，加重氧化损伤，可能是腺病毒潜伏感染参与慢性阻塞性肺病（COPD）发病的机制之一。

Objective To analyze the influence of adenovirus latent infection on gamma-glutamylcysteine systhetase（γ-GCS） in rat alveolar epithelial cells. Methods The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid. Glutathione（GSH） contents, the activity of γ-GCS were detected in oxidant stress. Then the leuel of protein expression, mRNA expression, and promoter transcriptional activity of glutamate-cysteine ligase catalytic subunit （GCLC） were further detected. Results GSH contents decreased because of adenovirus E1A expression in oxidant stress. E1A repressed the expression and activity of γ-GCS, messenger RNA expression, and promoter transcriptional activity of GCLC. Conclusion Adenovirus E1A decreased the activity of γ-GCS probably by repressed promoter transcriptional activity of GCLC. As a result, GSH contents were downregulated in oxidant stress. Thus Adenovirus latent infection amplified the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress, which may be an important mechanism of COPD.