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目的研究Pten基因对抗氧化蛋白Cu/Zn-SOD基因表达的调控,初步探索其调控机制。方法运用North-ern blot比较Pten+/+ MEFs与Pten-/- MEFs细胞中基础Cu/Zn-SOD mRNA表达差异,以及0.1mmol/LH2O2诱导后Pten+/+ MEFs与Pten-/- MEFs细胞中Cu/Zn-SOD mRNA表达差异;运用Western blot分析PI3K/AKT通路抑制剂LY294002作用Pten-/- MEFs细胞不同时间(30min,1、2、6、24h)后,与空白Pten-/- MEFs细胞相比,磷酸化AKT及Cu/Zn-SOD蛋白表达变化;利用Northern blot分析LY294002作用Pten-/- MEFs细胞30min,1、2、6、24h后,与空白Pten-/- MEFs细胞相比,Cu/Zn-SOD mRNA表达变化。结果Pten-/- MEFs细胞中,基础Cu/Zn-SOD mRNA表达水平降低,H2O2诱导的Cu/Zn-SOD mRNA表达明显受到抑制;在LY294002作用Pten-/- MEFs细胞30min时,AKT磷酸化水平明显降低,2h时基本没有表达;与此对应,在LY294002作用Pten-/- MEFs细胞30min时,Cu/Zn-SOD蛋白及mRNA表达均增强,并持续增强至24h。结论小鼠胚胎成纤维细胞中,Pten基因可通过拮抗PI3K/AKT信号通路调控Cu/Zn-SOD蛋白及mRNA的表达。
Objective To study the regulation of Pten gene on the expression of antioxidant protein Cu / Zn-SOD gene and its regulatory mechanism. Methods The difference of basic Cu / Zn-SOD mRNA expression in Pten + / + MEFs and Pten - / - MEFs cells was analyzed by North-ern blot and the effect of Cu / Zn-SOD mRNA expression differences; Pten - / - MEFs cells PI3K / AKT pathway inhibitor LY294002 Western blot analysis of different time (30min, 1,2,6,24h), compared with blank Pten - / - MEFs cells , Phosphorylated AKT and Cu / Zn-SOD protein expression; Northern blot analysis of LY294002 Pten - / - MEFs cells after 30min, 1,2,6,24h, compared with blank Pten - / - MEFs cells, Cu / Zn-SOD mRNA expression changes. Results The expression of Cu / Zn-SOD mRNA decreased and the expression of Cu / Zn-SOD mRNA induced by H2O2 was significantly inhibited in Pten - / - MEFs. The phosphorylation of AKT at 30 min after LY294002 treatment of Pten - / - MEFs The expression of Cu / Zn-SOD protein and mRNA in Pten - / - MEFs increased with LY294002 treatment for 30min, and continued to increase until 24h. Conclusion Pten gene can regulate the expression of Cu / Zn-SOD protein and mRNA by antagonizing the PI3K / AKT signaling pathway in mouse embryonic fibroblasts.