蛋白酶体活性对小鼠神经干细胞活性氧水平的影响

目的:探讨蛋白酶体活性对小鼠神经干细胞(NSCs)活性氧(ROS)水平和增殖能力的影响,寻求维持NSCs活力的有效方法。方法:分离培养新生(P0)和成年(P90)小鼠室管膜下区(SVZ)神经干细胞。比较P0和P90 NSCs成球数量和增殖能力,荧光酶标仪检测蛋白酶体活性,DCFH-DA法测定ROS水平。应用蛋白酶体抑制剂MG132和激活剂18α-GA分别作用于P0和P90 NSCs,CCK-8法、DCFH-DA法和JC-1染色检测蛋白酶体活性改变对NSCs增殖能力、ROS水平和线粒体膜电位的影响。结果:P90 NSCs神经球直径和数量较P0明显减少,增殖活性较P0 NSCs明显下降(P0.001)。此外,P90 NSCs蛋白酶体活性较P0 NSCs降低0.55±0.03(P0.05),但ROS水平却较P0 NSCs升高13.25±0.12倍(P0.001)。MG132作用后P0 NSCs ROS水平呈浓度依赖性升高,其中10μmol/L组较对照组显著升高131%±8.4%(P0.001),增殖活性却降低54.4%±7.8%(P0.001);MG132组NSCs线粒体膜电位较对照组下降。相反,18α-GA作用后P90 NSCs ROS水平呈浓度依赖性降低,其中6和8μg/ml组分别下降2.77±0.20和2.78±0.32(P0.01),增殖活性却是对照组的3.76和5倍(P0.001);18α-GA组NSCs线粒体膜电位较对照组升高。结论:蛋白酶体活性与NSCs ROS水平密切相关,激活蛋白酶体活性可减少ROS对成年NSCs线粒体功能的影响,提高NSCs增殖活力。

Effect of proteasomal activity on reactive oxygen species of neural stem cells in mice

Objective:To investigate the effect of proteasomal activity on reactive oxygen species (ROS) and proliferative ability of neural stem cells (NSCs) in mice,thus providing a way to maintain NSCs vitality.Methods:NSCs were dissociated from mice subventricular zone (SVZ) at postnatal day 0 (P0) and postnatal day 90 (P90).The neurosphere number and proliferation ability of NSCs were merasured.Proteasomal activity and ROS level were detected by fluorescence spectrophotometry and DCFH-DA,respectively.CCK-8,DCFH-DA and JC-1 staining were used to evaluate the change of proliferative ability,ROS levels and mitochondrial membrane potential of NSCs following treatment of P0 and P90 NSCs with proteasomal inhibitor MG132 and activator 18α-GA.Results:Accompanied with dramatically declined neurosphere number and proliferational activity in P90 NSCs (P ＜ 0.001),the proteasomal activity was decreased by 0.55 ±0.31 (P ＜0.05),but the ROS level was rised by 13.25 ±0.12 times (P ＜0.001) compared with P0 NSCs.The ROS levels were increased gradually in P0 NSCs treated by MG132 with different concentrations,charactered by elevated ROS level by 131％ ± 8.4％ (P ＜ 0.001) but decreased proliferational activity by 54.4％ ± 7.8％ (P ＜ 0.001) and reduced mitochondrial membrane potential in 10 μmol/L MG132 group compared with control group.In contrast,the ROS levels were declined gradually in P90 NSCs treated by 18α-GA with different concentrations,charactered by reduced ROS level by 2.77 ± 0.20 and 2.78 ± 0.32 times (P ＜ 0.01) but rised proliferational activity (P ＜ 0.001) and elevated mitochondrial membrane potential in 6 and 8 μg/rnl 18α-GA group compared with control group.Conclusion:Proteasome activity is closely related to ROS level in NSCs.Activation of proteasome activity can reduce the effect of ROS on mitochondrial function of adult NSCs and improve the self-renewal ability of NSCs.