神经干细胞衰老与核因子E2相关因子2/抗氧化反应元件信号通路的关系

Relationship between neural stem cells aging and nuclear factor erythroid 2 related factor 2/antioxidant response element signaling pathway

目的建立D-半乳糖(D-gal)诱导小鼠神经干细胞(NSCs)体外衰老模型,探讨NSCs衰老与细胞核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路的关系。方法取新生C57BL/6J小鼠全脑,分离、鉴定NSCs,培养至第3代,随机分为对照组和衰老组。对照组细胞在NSCs培养基中常规培养48 h,衰老组细胞在对照组基础上加入D-gal(终浓度为10 g/L)。细胞计数试剂盒8(CCK-8)法检测NSCs增殖活力;衰老相关β-半乳糖苷酶(SA-β-gal)染色检测衰老阳性神经球百分率;锥虫蓝染色检测细胞存活率;酶标比色法检测细胞培养上清液中超氧化物歧化酶(SOD)与过氧化氢酶(CAT)活性和丙二醛(MDA)含量;Western boltting检测NSCs中Nrf2和血红素加氧酶1(HO-1)蛋白表达水平;Real-time PCR检测GCLC和GCLM基因表达水平。结果与对照组相比,衰老组NSCs增殖活力明显下降,锥虫蓝染色显示,NSCs存活率下降明显,SA-β-gal染色阳性神经球百分率显著上升,SOD活性与CAT活性均显著降低,MDA含量显著升高,NSCs中Nrf2/ARE信号通路相关蛋白Nrf2和HO-1表达水平显著下调,通路相关GCLC和GCLM基因表达下调。结论采用D-gal可以构建NSCs体外衰老模型,其氧化损伤机制可能与抑制Nrf2/ARE抗氧化信号通路有关。

Objective To establish aging model of mouse neural stem cells( NSCs) in vitro and study the relationship between D-galactose( D-gal)-induced NSCs aging and nuclear factor erythroid 2 related factor 2( Nrf2)/antioxidant response elemen( ARE) signaling pathway. Methods The whole brain of newborn C57 BL/6 J mouse was obtained. NSCs were isolated and identified,and cultured the cells to the third generation. The NSCs were randomly divided into control group and aging group. The NSCs of control group were cultured in NSCs medium for 48 hours,and the NSCs of aging group were added with D-galactose( D-gal,final concentration of 10 g/L) on the basis of the control group.Cell counting kit-8( CCK-8) was used to detect the activity of NSCs;Senescence-associated β-galactosidase( SA-β-gal)staining was used to detect the percentage of senescence-positive neurospheres;Trypan blue staining was used to detect cell viability;Enzyme-labeled colorimetry assay was used for detection of the activity of superoxide dismutases( SOD),catalase( CAT) and malondialdehyde( MDA) in the cell culture supernatants;Western blotting was used to detect the expression of Nrf2,heme oxygenase-1( HO-1) protein;Real-time PCR was applied to detect the expression level of GCLC,GCLM genes. Results Compared with the control group,the proliferative activity of NSCs in the aging group decreased significantly,the percentage of SA-β-gal staining positive neurons increased obviously,the activity of trypan blue stained NSCs decreased explicitly,SOD activity and CAT activity decreased markedly,and MDA content increased significantly.The expression levels of Nrf2/ARE signaling pathway-related proteins Nrf2 and HO-1 were significantly down-regulated in cells,the expression of pathway-associated GCLC and GCLM genes was down-regulated. Conclusion The addition of Dgal to the culture system can construct an in vitro aging model of NSCs. The possible mechanism is closely related to the inhibition of the antioxidant activity of Nrf2/ARE signaling pathway.