【摘要】：目的:探討蛋白酶體活性對小鼠神經(jīng)干細(xì)胞(NSCs)活性氧(ROS)水平和增殖能力的影響,尋求維持NSCs活力的有效方法。方法:分離培養(yǎng)新生(P0)和成年(P90)小鼠室管膜下區(qū)(SVZ)神經(jīng)干細(xì)胞。比較P0和P90 NSCs成球數(shù)量和增殖能力,熒光酶標(biāo)儀檢測蛋白酶體活性,DCFH-DA法測定ROS水平。應(yīng)用蛋白酶體抑制劑MG132和激活劑18α-GA分別作用于P0和P90 NSCs,CCK-8法、DCFH-DA法和JC-1染色檢測蛋白酶體活性改變對NSCs增殖能力、ROS水平和線粒體膜電位的影響。結(jié)果:P90 NSCs神經(jīng)球直徑和數(shù)量較P0明顯減少,增殖活性較P0 NSCs明顯下降(P0.001)。此外,P90 NSCs蛋白酶體活性較P0 NSCs降低0.55±0.03(P0.05),但ROS水平卻較P0 NSCs升高13.25±0.12倍(P0.001)。MG132作用后P0 NSCs ROS水平呈濃度依賴性升高,其中10μmol/L組較對照組顯著升高131%±8.4%(P0.001),增殖活性卻降低54.4%±7.8%(P0.001);MG132組NSCs線粒體膜電位較對照組下降。相反,18α-GA作用后P90 NSCs ROS水平呈濃度依賴性降低,其中6和8μg/ml組分別下降2.77±0.20和2.78±0.32(P0.01),增殖活性卻是對照組的3.76和5倍(P0.001);18α-GA組NSCs線粒體膜電位較對照組升高。結(jié)論:蛋白酶體活性與NSCs ROS水平密切相關(guān),激活蛋白酶體活性可減少ROS對成年NSCs線粒體功能的影響,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 偽-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 鹵0. 03 compared with P0 NSCs (P0.05), but the level of ROS increased 13. 25 鹵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% 鹵8.4% (P0.001), but decreased the proliferation activity by 54.4% 鹵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 偽-GA, in which 6 渭 g/ml and 8 渭 g/ml decreased 2.77 鹵0.20 and 2.78 鹵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 偽 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.
【作者單位】： 山西醫(yī)科大學(xué)人體解剖學(xué)教研室;山西醫(yī)科大學(xué)第一臨床醫(yī)學(xué)系;
【基金】：山西省自然科學(xué)基金(2015011132) 山西省歸國留學(xué)人員基金(2014-033) 山西醫(yī)科大學(xué)大學(xué)生創(chuàng)新創(chuàng)業(yè)校級項(xiàng)目(20160108)
【分類號】：R741

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