异氟烷通过激活Calpain影响大鼠海马神经干细胞的增殖与分化

Isoflurane Affects Proliferation and Differentiation of Neural Stem Cells by Activating Calpain

目的探讨异氟烷影响神经干细胞(NSCs)增殖与分化的机制。方法原代培养SD新生大鼠(出生1d内)海马NSCs,取传至第2代细胞,将其随机分为4组(n=5):空白对照组(CON),给予含有21%O2和5%CO2的混合气体;异氟烷组(ISO),给予3.4%异氟烷,干预6h;Calpeptin组(CP),给予10μmol/L Calpeptin和含有21%O2和5%CO2的混合气体,干预6h;异氟烷+Calpeptin组(ISO+CP),给予10μmol/L Calpeptin和3.4%异氟烷,干预6h。BrdU检测NSCs增殖,Western blot检测GFAP、Tuj-1、αⅡ-spectrin及其裂解产物SBDP145的蛋白水平。结果与CON组相比,ISO组在干预后0、12、24h时BrdU+细胞数目明显减少,说明异氟烷抑制了NSCs增殖并有后遗效应;ISO组SBDP145表达明显上调,说明异氟烷可上调Calpain活性。掺入Calpain抑制剂Calpeptin后,与ISO组相比,ISO+CP组SBDP145表达量明显下降,表明Calpeptin可拮抗异氟烷致Calpain活性增强的作用;ISO+CP组BrdU+细胞数目增多,Calpeptin减弱了异氟烷抑制NSCs增殖的作用。与CON组相比,ISO组GFAP表达水平上调,Tuj-1水平无明显改变;CP组GFAP水平无明显改变,而Tuj-1表达上调。与ISO组相比,ISO+CP组GFAP水平降低,Tuj-1水平无明显改变,表明异氟烷促进NSCs分化为胶质细胞,而Calpeptin可拮抗异氟烷的这种作用。结论异氟烷通过激活Calpain信号通路抑制NSCs的自我更新并促进NSCs分化为胶质细胞。

Objective To investigate the mechanism by which isoflurane（ISO）affects the proliferation and differentiation of neural stem cells（NSCs）.Methods NSCs were isolated from neonatal Sprague Dawley rats（postnatal day 1）and cultured.NSCs of passage 2were used and divided into 4groups（n=5）：control group,in which NSCs were treated with 5%CO2and 21%O2for 6h;ISO group,in which 3.4%ISO was given for intervention for 6h;calpeptin（CP）group,in which NSCs were treated with10μmol/L CP and 5%CO2,21%O2for 6h;ISO＋CP group,in which 10μmol/L CP and 3.4% ISO were given for 6h.The proliferation of NSCs was detected by using 5-bromodeoxyuridine（BrdU）.The expression levels of GFAP,Tuj-1,αⅡ-spectrin and SBDP145 were determined by Western blot.Results The number of BrdU＋cells was significantly decreased at 0,12,24 h in ISO group when compared with control group,suggesting that ISO can inhibit the proliferation of NSCs.The expression of SBDP145 was conspicuously upregulated in ISO group,indicating that ISO can increase the activity of calpain.After treatment with CP,the expression of SBDP145 was markedly decreased in ISO＋CP group when compared with ISO group,suggesting that CP can suppress the ISO-induced increase of calpain activity.The number of BrdU＋cells was increased in ISO＋CP group,suggesting that CP could block antiproliferative effect of ISO on NSCs.The expression levels of GFAP were significantly increased in ISO group when compared with control group and there was no significant difference in Tuj-1expression between the two groups.Be contrast,cells in CP group experienced no significant change of GFAP but significant upregulation of Tuj-1.The expression level of GFAP was reduced while Tuj-1had no significant change in ISO＋CP group when compared with ISO group,which suggested that ISO can promote the differentiation of NSCs to gliocytes and such ISO-induced effect could be blocked by CP.Conclusion ISO inhibits the self-renewal capacity of NSCs and promotes the differentiation of NSCs to gliocytes by activating calpain signaling pathways.