蛋白酶体活性对小鼠神经干细胞活性氧水平的影响

发布时间：2018-10-24 12:06

【摘要】：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。
[Abstract]:Aim: to investigate the effect of proteasome activity on the level and proliferation of (NSCs) reactive oxygen species (Ros) in mouse neural stem cells (NSCs), and to find an effective way to maintain the activity of NSCs. Methods: (SVZ) neural stem cells in subependymal region of newborn (P 0) and adult (P 90) mice were isolated and cultured. The number and proliferative ability of P0 and P90 NSCs were compared. The activity of proteasome was detected by fluorescence enzyme marker and the level of ROS was measured by DCFH-DA. Proteasome inhibitor MG132 and activator 18 伪-GA were applied to P0 and P90 NSCs,CCK-8 methods respectively. The effects of proteasome activity on NSCs proliferation, ROS level and mitochondrial membrane potential were detected by DCFH-DA and JC-1 staining. Results: the diameter and number of P90 NSCs neurospheres were significantly decreased than that of P0, and the proliferative activity was significantly lower than that of P0 NSCs (P0. 001). In addition, the activity of P90 NSCs proteasome decreased by 0. 55 卤0. 03 compared with P0 NSCs (P0.05), but the level of ROS increased 13. 25 卤0. 12 times than that of P0 NSCs (P0. 001). The level of P0 NSCs ROS increased in a concentration-dependent manner after MG132 treatment. Compared with the control group, 10 渭 mol/L group significantly increased 13.1% 卤8.4% (P0.001), but decreased the proliferation activity by 54.4% 卤7.8% (P0.001), and the mitochondrial membrane potential of NSCs in the MG132 group was lower than that in the control group. On the contrary, the level of P90 NSCs ROS decreased in a concentration-dependent manner after treatment with 18 伪-GA, in which 6 渭 g/ml and 8 渭 g/ml decreased 2.77 卤0.20 and 2.78 卤0.32 (P0.01), respectively, but the proliferative activity was 3.76 and 5 times higher than that in the control group, and the mitochondrial membrane potential of NSCs in 18 伪 GA group was higher than that in the control group. Conclusion: the activity of proteasome is closely related to the level of NSCs ROS. Activation of proteasome activity can reduce the effect of ROS on mitochondrial function of adult NSCs and increase the proliferative activity of NSCs.
【作者单位】：山西医科大学人体解剖学教研室;山西医科大学第一临床医学系;
【基金】：山西省自然科学基金(2015011132) 山西省归国留学人员基金(2014-033) 山西医科大学大学生创新创业校级项目(20160108)
【分类号】：R741

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