自然衰老小鼠室管膜下区神经干细胞增龄性改变

Age-related changes of neural stem cells from the subventricular zone of aged mice

目的探讨自然衰老小鼠室管膜下区(SVZ)神经干细胞(NSCs)在衰老过程中生物学特点的改变。方法 2月龄、28月龄小鼠,各10只,体外分离、培养、鉴定NSCs,5-溴-2-脱氧尿苷(Brd U)染色比较两组细胞的增殖水平;β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老水平;细胞内活性氧(ROS)试剂盒检测细胞ROS表达水平;Western blotting检测细胞周期蛋白D1(cyclin D1)、p16及p19蛋白表达水平;体内用巢蛋白(Nestin)/性别决定基因高迁移率组蛋白-2(Sox2)检测两组小鼠SVZ区厚度的差异。结果年轻、年老小鼠的神经干细胞能表达Nestin及Sox2,符合神经干细胞的表达特性。与年轻小鼠相比,年老小鼠神经干细胞的Brd U阳性细胞比例显著下降,SA-β-Gal阳性细胞百分比显著升高,细胞活性氧水平显著升高,SVZ区厚度明显变薄,差异具有统计学意义(P<0.05)。在分子水平上表现为cyclin D1蛋白表达水平明显下降,p16、p19蛋白表达水平显著升高。结论年老小鼠NSCs出现增龄性变化,这些变化可能在大脑衰老中起重要作用。

Objective To investigate the biological changes of neural stem cells（ NSCs） of mice from subventricular zone（ SVZ） during aging. Methods NSCs were isolated,cultured and identified from 2 month（ young）and 28 month（ aged） old mice respectively,with 10 mice in each group. The proliferation ability of NSCs was determined by Brd U staining; cellular senescence was detected by senescence-associated-β-galactosidase（ SA-β-gal） staining; ROS level was determined by ROS kit; expression of cyclin D1,p16,p19 protein in NSCs was tested by Western blotting. The thickness of SVZ between the two groups was detected by nestin / Sox2 staining. Results Positive expressions of neural stem cell marker nestin and Sox2 were identified in NSCs of the young and the aged mice. Aged NSCs exhibited a decreased percentage of Brd U positive cells, increased SA-β-gal activity, elevated intracellular reactive oxygen species level,decreased pool size in the SVZ zone,and there were significant differences（ P〈0. 05）. Additionally,at the molecular level,aged NSCs exhibited a decrease of cyclin D1 and an increase of p16 and p19. Conclusion Aged NSCs demonstrate aging changes which may play an important role in brain aging.