增生期和分化期大鼠神经干细胞过氧化氢预处理氧化耐受的体外实验研究

目的探讨神经干细胞(NSCs)在增生期和分化期过氧化氢(H2O2)预处理后H2O2损伤氧化耐受的变化。方法分别用浓度为0.05 mmol/L、0.10 mmol/L、0.25 mmol/L、0.50 mmol/L、1.0 mmol/L的H2O2处理增生期NSC(PNSC)和分化期NSC(DNSC)。用0.50 mmol/L的H2O2处理10 m in后,间隔6 h和12 h后再次给予0.50 mmol/L H2O2,观察细胞和损伤的情况。检测热休克蛋白27(Hsp27)、热休克蛋白70(Hsp70)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的变化。透射电镜(TEM)检测NSC超微结构变化。结果经0.50 mmol/L H2O2预处理10 m in,间隔12 h后DNSC可以产生H2O2耐受,并至少持续12 h;而PNSC并不能产生H2O2耐受。预处理后的DNSC在Hsp27和Hsp70阳性细胞比例显著上升(P<0.05),TEM示存活的细胞均为比较原始的细胞。结论在给予0.5 mmol/L H2O2预处理10 m in后,间隔12 h后DNSC后产生对H2O2耐受;而PNSC不能产生耐受。这可能与Hsp27和Hsp70的增加有关。此外,这与原始状态NSC H2O2氧化耐受要高于分化状态的细胞有关。

Hydrogen Peroxide Preconditioning and Oxidative Tolerance of Neural Stem Cells of Rat in Vitro

Objective To evaluate the effects of preconditioning with hydrogen peroxide(H_2O_2) on oxidative tolerance(OT) of rat neural stem cells(NSC) in proliferation(PNSC) and differentiation(DNSC) respectively.Methods PNSC and DNSC were cultured in vitro.H_2O_2 was used in following concentrations: 0.05,0.10,0.25,0.50 and 1.0 mmol/L,for 2 h,4 h,8 h,12 h and 24 h respectively to assess the cell injury.Oxidative preconditioning was undertaken with final concentration of 0.50 mmol/L H_2O_2 for 10 min.After 6 h or 12 h preconditioning,0.50 mmol/L H_2O_2 were added to cause insult.Hsp27,Hsp70,catalase(CAT),superoxide dismutase(SOD) assessed by immunohistochemistry(IHC),viability of CAT and SOD were assessed before and 12 h after preconditioning.TEM was used to study the ultrastructure.Results When H_2O_2 reaches 0.25 mmol/L,both PNSC and DNSC develop injury.Twelve hours after 0.50 mmol/L H_2O_2 preconditioning for 10 min in DNSC,the cell injury decrease significantly than the non-preconditioning group.The preconditioning OT can last at lest for 12 h.However,PNSC do not develop OT.DNSC presented significant more cells with positive staining of Hsp27 and Hsp70;however both the staining and viability of CAT and SOD keep no significant difference before and after preconditioning(P<0.05).The TEM demonstrated after preconditioning,more cells keep alive during H_2O_2 insult,and all surviving present characteristics of immature cells.Conclusion Although DNSC develops OT after 0.50 mmol/L H_2O_2 preconditioning for 10 min with 12 h interval,and the OT last for 12 h,the PNSC can't develop.The mechanism may involve the protection of Hsp27,Hsp70,but not CAT nor SOD.In addition,immature NSC get higher OT than differentiated NSC.