18α-GA激活Nrf2对成体神经干细胞的影响

发布时间：2018-06-26 18:44

  本文选题：α-甘草次酸 + 成体神经干细胞　； 参考：《中国病理生理杂志》2017年10期

【摘要】：目的:明确18α-甘草次酸(18α-GA)对核因子E2相关因子2(Nrf2)的激活作用,观察上调Nrf2对成体神经干细胞(a NSCs)增殖和分化能力的影响,探讨维持a NSCs活力的有效途径。方法:体外培养新生、成年和老年小鼠的侧脑室室膜下区神经干细胞(NSCs),观察随年龄增长NSCs中Nrf2的表达变化。以18α-GA作用a NSCs,用real-time PCR和Western blot实验比较18α-GA组与DMSO对照组a NSCs的Nrf2表达变化。构建携带绿色荧光蛋白(GFP)基因的Nrf2-shRNA慢病毒载体(LV)感染a NSCs,用real-time PCR和Western blot实验检测shRNA对Nrf2的沉默效率。将a NSCs按照干预条件不同分为DMSO组、18α-GA组、LV-GFP组和LV-Nrf2-shRNA组,运用Brd U掺入实验、Tuj1免疫荧光染色、CCK-8实验、Hoechst 33342/PI双染色和活性氧簇(ROS)水平测定等方法评价18α-GA是否通过激活Nrf2对a NSCs的增殖、分化、细胞活力、细胞凋亡以及氧化应激水平产生影响。结果:随年龄增长,成年和老年小鼠a NSCs的Nrf2 mRNA表达水平较新生小鼠NSCs显著降低(P0.01),但a NSCs的ROS水平显著高于新生小鼠NSCs(P0.05)。应用18α-GA后,a NSCs的Nrf2 mRNA与蛋白表达水平较DMSO组明显升高(P0.01),并且a NSCs的Brd U阳性率也显著增加(P0.01)。2 mg/L 18α-GA作用后,a NSCs的Tuj1阳性率和细胞活力较DMSO组显著升高(P0.05),而凋亡率和ROS水平显著下降(P0.05和P0.01)。但是,敲减a NSCs的Nrf2并应用18α-GA干预后,LV-Nrf2-shRNA组Brd U阳性率、Tuj1阳性率以及细胞活力均较LV-GFP组显著下降(P0.05),ROS水平却显著上升(P0.05)。结论:18α-GA可能通过激活Nrf2提高a NSCs的抗氧化能力,促进a NSCs增殖和分化,维持a NSCs潜能。
[Abstract]:Aim: to investigate the activation of 18 伪 -glycyrrhetinic acid (18 伪 -GA) on the nuclear factor E2 related factor 2 (Nrf2), to observe the effect of Nrf2 on the proliferation and differentiation of adult neural stem cells (a NSCs), and to explore an effective way to maintain the activity of a NSCs. Methods: neural stem cells (NSCs) in the submembranous region of lateral ventricle of newborn, adult and aged mice were cultured in vitro, and the expression of Nrf2 in NSCs was observed with age. The expression of nrf2 in 18 伪 -GA group was compared with that in DMSO control group by real-time PCR and Western blot. Nrf2-shRNA lentivirus vector (LV) carrying green fluorescent protein (GFP) gene was constructed to infect a NSCs.The silencing efficiency of shRNA to Nrf2 was detected by real-time PCR and Western blot assay. A NSCs were divided into LV-GFP group and LV-Nrf2-shRNA group in DMSO group according to different intervention conditions. The proliferation of a NSCs was evaluated by Brd U incorporation assay (Brd U incorporation) and Hoechst 33342% Pi double staining and reactive oxygen species cluster (Ros) level. Differentiation, cell viability, apoptosis and oxidative stress levels have an effect. Results: the expression of Nrf2 mRNA in adult and aged mice was significantly lower than that in newborn NSCs (P0.01), but the Ros level of a NSCs was significantly higher than that of NSCs in newborn mice (P0.05). The expression of Nrf2 mRNA and protein in NSCs was significantly higher than that in DMSO group (P0.01), and the positive rate of BrdU in a-NSCs was also significantly increased (P0.01). 2 mg / L 18 伪 -GA treatment, the Tuj1 positive rate and cell viability of NSCs were significantly higher than those of DMSO group (P0.05), while the apoptosis rate and apoptosis rate of NSCs were significantly higher than those of DMSO group (P0.05). Ros level decreased significantly (P0.05 and P0.01). However, the positive rate of Brd-U and cell viability in LV-Nrf2-shRNA group were significantly lower than those in LV-GFP group (P0.05) after Nrf2 knockout and 18 伪 -GA intervention (P0.05). Conclusion it is suggested that the activation of Nrf2 may increase the antioxidant capacity of a NSCs, promote the proliferation and differentiation of a NSCs, and maintain the potential of a NSCs.
【作者单位】： 山西医科大学人体解剖学教研室;山西医科大学生物制药系;
【基金】：国家自然科学基金资助项目(No.81571381) 山西省自然科学基金资助项目(No.2015011132) 山西医科大学校级博士启动基金(No.03201604)
【分类号】：R329.2

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