Akt/mTOR信号通路介导低浓度过氧化氢对小鼠表皮干细胞的促增殖作用

Akt/m TOR pathway mediates the proliferation of murine epidermal stem cells following low concentration of hydrogen peroxide treatment

目的:探讨低浓度过氧化氢(H_2O_2)对小鼠表皮干细胞(mESCs)体外促增殖作用及其机制。方法:以酶消化法分离培养mESCs。免疫荧光技术鉴定第3代mESCsβ1整合素和角蛋白19(K19)表达,流式细胞术鉴定CD34的表达;以不同低浓度H_2O_2(0、25、50、100、150和200μmol/L)处理mESCs 48 h和72h。同时,另一组实验中在用不同浓度H_2O_2刺激的同时加入或不加入雷帕霉素(50 ng/ml)培养48 h。甲氮甲唑蓝(MTT)法检测细胞增殖;Western Blot法检测H_2O_2刺激小鼠mESCs 48 h Akt/mTOR信号通路关键蛋白mTOR、Akt、P70s6k、e IF4E、4E-BP1、p-mTOR、p-Akt、p-P70s6k、p-eIF4E、p-4E-BP1和细胞增殖相关蛋白Cyclin D1、增殖细胞核抗原(PCNA)的表达。结果:免疫荧光技术检测分离培养的mESCs细胞100%表达β1整合素和K19;流式细胞术检测99.8%的mESCs表达CD34;MTT检测结果显示,48 h和72 h时25μmol/L和50μmol/L的H_2O_2可以有效地促进mESCs的增殖(P<0.05),而雷帕霉素能拮抗这一效应;但当H_2O_2浓度为200μmol/L时,则显著抑制mESCs增殖(P<0.05);免疫印迹结果显示低浓度H_2O_2处理mESCs 48 h后,Akt/mTOR信号通路关键蛋白mTOR、Akt、eIF4E、4E-BP1水平及其磷酸化水平(p-mTOR、p-eIF4E、p-4E-BP1)显著增加(P<0.05或P<0.01),当H_2O_2浓度为200μmol/L时则抑制其表达(P<0.05或P<0.01)。此外,50μmol/L的H_2O_2能上调细胞周期蛋白Cyclin D1和PCNA的表达,而雷帕霉素能拮抗这一作用。结论:低浓度的H_2O_2对mESCs有明显的促增殖作用,并且能激活Akt/mTOR通路。而低浓度H_2O_2对mESCs的促增殖作用至少部分是通过活化Akt/mTOR信号通路介导的。

Objective:To investigate the effect of a low concentration of hydrogen peroxide(H_2O_2) on the proliferation of murine epidermal stem cells(mESCs) and its mechanism. Methods: The mESCs were isolated with trypsin and type I collagen enzyme digestion. The mESCs were identified using β1 integrin, keratin 19(K19) and CD34 antibody with immunofluorescence detection technique and flow cytometry analysis. The mESCs were treated with different concentrations of H_2O_2(0, 25, 50, 100, 150 and 200 μmol/L) for 48 hours and 72 hours, and in another experiment, the mESCs were treated with low concentrations of H_2O_2(0, 25, 50, 100, 150 μmol/L) alone or combined with 50 ng/ml rapamycin(Rapa) for 48 hours. The proliferation of mESCs was detected by MTT analysis, and the expression of key proteins such as mTOR, Akt, P70s6 k, eIF4E,4E-BP1, p-Akt, p-mTOR, p-P70s6 k,p-4EBP1 and p-eIF4E in the Akt/mTOR signaling pathway was assayed using Western blotting after mESCs treated with different concentrations of H_2O_2 for 48 hours. The expression of cell cycle protein cyclin D1 and proliferating cell nuclear antigen(PCNA) were also determined using Western blotting. Results: The results showed that 100% of the mESCs expressed β1 integrin and K19, and 99.8% of them expressed CD34, thereby mESCs were comfirmed. The proliferation of mESCs was makedly increased 48 and 72 hours after H_2O_2 treatment at the dose of 25 μmol/L and 50 μmol/L(P <0.05), and this proliferation effect as induced by H_2O_2 was antagonized by rapamycin. However, the proliferation of mESCs was inhibited markedly after treatment with 200 μmol/L H_2O_2 for 48 hours and 72 hours(P <0.05), Furthermore, after mESCs were treated with H_2O_2 in a concentration of 25 μmol/L to 150 μmol/L, the Akt/mTOR pathway was activated, characterized by a marked increase of expression of total and phosphorylated key proteins( p-mTOR, p-4E-BP1 and p-eIF4E) in this pathway(P <0.05 or P <0.01), but the expression of these proteins was dramatically attenuated after being treated with 200 μmol/L of H_2O_2(P <0.05 or P <0.01). In addition, the expression of cell cycle protein cyclin D1 and PCNA were increased(P<0.05) after being treated with 50 μmol/L H_2O_2, and it was antagonized by treatment of rapamycin. Conclusions: Low concentration of H_2O_2 can promote the proliferation of mESCs, and it activates the Akt/mTOR pathway. The mESCs proliferation induced by low concentration H_2O_2 is partly dependant on the Akt/mTOR pathway activation.