H2O2及PTEN在肽类生长因子活化Akt激酶中的作用

Effects of H_2O_2 and PTEN on activation of Akt by peptide growth factors

目的研究H2O2、PTEN与肽类生长因子(胰岛素等)在调控Akt激酶活性中的作用及其相互关系。方法Western Blot检测对照小鼠胚胎成纤维细胞PTEN+/+ MEFs和PTEN基因敲除小鼠胚胎成纤维细胞PTEN-/-MEFs中PTEN蛋白表达的差异。用胰岛素及NADPH氧化酶(NOX)抑制剂——DPI作用PTEN+/+M EFs和PTEN-/-MEFs细胞,Western Blot检测Akt激酶蛋白磷酸化水平的变化。将细胞接种于96孔板,加入胰岛素及DPI,96h后MTT法检测细胞增殖的变化。结果在对照PTEN+/+ MEFs细胞内,加入胰岛素(100mU/ml)15min后Akt激酶磷酸化水平明显上调,加入10μmol/LDPI处理30min后,Akt激酶磷酸化恢复到基础水平,在PTEN-/-MEFs细胞中未观察到上述变化。经胰岛素作用后PTEN+/+ MEFs细胞增殖加快(P<0.05),加入DPI作用后可减缓细胞的增殖(P<0.05),胰岛素和DPI同时作用对其增殖无明显影响(P>0.05);各种处理因素对PTEN-/-MEFs细胞增殖均无明显影响(P均>0.05)。结论肽类生长因子可以通过磷酸化激活Akt激酶,这一过程依赖于H2O2的产生和抑癌基因PTEN的存在。

Objective To research the role and the interrelationship of peptide growth factor （insulin）, H2O2 and PTEN in the regulation to the Akt kinase. Method The expression of PTEN protein in control immortalized wild-type mouse embryonic fibroblast cells （PTEN MEFs） and PTENnull cells （PTEN METs） was determined by Western blot. The two types of cells were treated with insulin and/or diphenyleneiodonium （DPI）, and then the phosphorylation level of Akt kinase was detected by Western blot. The two types of cells were placed in 96-well cell culture plates with the treatments of insulin and/or DPI for 96 hours, then the proliferation was assessed by MTT assay. Result In the control cells of PTEN MEFs, the phosphorylation level of Akt kinase was up-regulated obviously when treated with insulin （100mU/mI） for 15min, while it was come back to the basal level after the addition of DPI 10μmol/L for 30min. However these influences were not observed in PTEN MEFs cells. Treatment with insulin could facilitated the proliferation of the control cells of PTEN＇ MEFs （P〈0.05）, while the proliferation were depressed after DPI were given （ P〈0.05）. Furthermore, when the control cells PTEN MEFs treated with the insulin and the DPI at the same time, the proliferation changed weakly （ P〉0. 05）. However neither of the treatments could affect the proliferation of the PTEN MEFs cells obviously （ P〉0. 05）. Conclusion The peptide growth factors can activate the Akt kinase by phosphorylation. This process depends on the production of H2O2 and the presence of PTEN.