体外神经干细胞培养过程中蛋白激酶磷脂酰肌醇-3激酶/AKT信号途径的作用

Role of the phosphatidylinositol 3-kinase/AKT signaling pathway in culturing neural stem cells in vitro

目的:探讨磷脂酰肌醇-3激酶(phosphatidylinositol-3kinase,PI3K)/AKT信号途径在体外培养神经干细胞存活、分化中的作用。方法:实验于2001-11/2004-04在重庆医科大学神经病学研究所完成。分为对照组、PI3K特异性抑制剂wortmannin组和LY294002组;采用间接免疫荧光法检测nestin,NF-200和GFAP的表达;TUNEL评价细胞凋亡率;WesternBlot法检测磷酸化的AKT,caspase-9和bad的表达。结果:随着wortmannin和LY294002浓度增大,神经干细胞的形态变化逐渐明显,而且细胞凋亡率也逐渐增加。低浓度时(wortmannin浓度为5nmol/L,LY294002浓度为2μmol/L),神经干细胞凋亡率与对照组无显著性意义(P>0.05);高浓度时(wortmannin浓度为50,100nmol/L,LY294002浓度为50,100μmol/L),神经干细胞凋亡率与对照组、低浓度组有非常显著性意义(P<0.01)。WesternBlot结果提示,随着PI3K特异性抑制剂浓度增大,磷酸化AKT的表达逐渐减弱。当高浓度wort-mannin(20nmol/L)和LY294002(10μmol/L)抑制了AKT磷酸化后,磷酸化的caspase-9,Bad表达减弱。此外,将wortmannin组和LY294002组存活的细胞接种后,分化后细胞的NF-200,GFAP表达无显著性意义。结论:神经干细胞存活依赖于PI3K/AKT途径的活化,其机制可能是PI3K/AKT活化后。

AIM:To explore the role of the phosphatidylinositol 3 kinase(PI3K) /AKT signaling pathway in the survival and differentiation of neural stem cells(NSCs)in vitro. METHODS:The experiment was finished in Clinical Neurology Institut,Chongqing University of Medical Sciences from November 2001 to April 2004.NSCs were randomly divided into control group,wortmannin group and LY294002 group.Wortmannin and LY294002 are the distinctive inhibitors of PI3K.The expressions of nestin,NF 200 and GFAP were detected with indirect immunofluorescence.The ratio of cell apoptosis was estimated with TUNEL;the expressions of phosphorylated AKT,caspase 9 and bad were detected with Western blot. RESULTS:With the increase of concentration of wortmannin and LY294002,the morphologic change of NSCs was distinct gradually and the apoptoticrate increased gradually.The apoptotic rate of NSCs had no obvious difference at low concentration(5 nmol/L for wortmannin,2 μmol/L for LY294002) compared with the control group(P >0.05).There was significant differences at high concentration (50,100 nmol/L for wortmannin;50,100 μmol/L for LY294002) compared with the control group and lower concentration group(P< 0.01).The results of Western blot indicated that the expression of phosphorylated AKT decreased gradually when the correntrations of wortmannin and LY294002 increased.But when high concentration of wortmannin(20 nmol/L) and LY294002(10 μmol/L) inhibited AKT acidification.,the expression of phosphorylated caspase 9 and bad decreased.After the survival cells of wortmannin and LY294002 groups were inoculated,neurofilament protein(NF) 200 and glial fibrillary acidic protein(GFAP) of differentiating cells had no significant changes. CONCLUSION:The survival of NSCs depended on the activation of PI3 K/AKT pathway.It may accelerate phosphorylation of bad and caspase 9 and inhibit the occurrence of apoptosis.But PI3 K/AKT pathway may have little function in the differentiation of NSCs.