活性氧（ROS）对神经干细胞增殖影响及其机制研究

发布时间：2018-05-24 03:12

本文选题：神经干细胞 + 活性氧　；参考：《南京医科大学》2014年硕士论文

【摘要】：神经干细胞(NSCs),作为潜在的神经退行性疾病及中枢损伤修复的治疗方法,其增殖过程受多种因素调控,活性氧(ROS)即为其中之一。目前的研究结果显示不致于引起氧化应激的低浓度ROS可作为细胞内信号转导分子,对NSCs的增殖具有促进作用。B细胞特异性莫洛尼白血病病毒插入位点1(Bmi1)基因是多梳家族中重要的转录抑制性基因,可通过负性调控P16和P19在NSCs增殖和自我更新过程中发挥重要作用。不仅如此,Bmi1可调控线粒体功能进而调控ROS水平以维持细胞内氧化还原稳态。Bmi1半剂量缺失(Bmi1+/-)可下调NSCs内P16和P19,但NSCs的增殖能力与野生型(WT)无显著性差异,导致这种现象的内在机制尚不清楚。我们推测Bmi1表达下调所导致的ROS水平轻微上调,促进了NSCs的增殖,抵消了因Bmi1基因下调触发的细胞周期蛋白调控异常而产生的NSCs增殖阻滞效应。为了验证此推测,本学位论文对源于WT和Bmi1+/-新生小鼠脑中的NSCs分别进行体外培养,并给予不同浓度的H202和抗氧化剂NAC处理调控胞内ROS水平,应用神经球分析法、MTT和BrdU试验、胞内ROS检测等方法,探究ROS和Bmi-1对NSCs增殖的影响以及其相互作用机制。结果显示,1)在基础状态下,Bmi1+/--NSCs与WT-NSCs的增殖及自更新能力并没有显著差异,但胞内ROS水平轻度增加。2)经1 μM H2O2处理24 h后,WT-NSCs胞内ROS水平轻度上调,与此一致,神经球数量也略有增加,NSCs的细胞活力显著增强,BrdU插入比率亦有增加,虽然神经球的直径无明显变化,并且总抗氧化能力(T-AOC)和总超氧化物岐化酶活性(T-SOD)增高；Bmi1+/--NSCs胞内ROS增加较WT-NSCs更为显著,但上述其他指标均较基础状态下降,并与WT-NSCs存在显著性差异。经10 μM H2O2处理24 h后,Bmil+/--NSCs胞内ROS较WT-NSCs增加更为显著,上述指标在两种基因型NSCs均下降,但Bmmi1+/-组更显著。经100 μM H2O2处理24 h后,两种基因型NSCs胞内ROS水平均显著降低,并且上述其他指标均进一步显著下调,除Bmil+/--NSCs的BrdU插入比率低于VT-NSCs外,两种基因型之间的差异也不明显。3)在经不同浓度(1、5和10 mM)的ROS清除剂NAC处理24 h后,WT-NSCs胞内ROS水平、神经球的数量、直径及细胞活力、BrdU插入比率以及T-AOC和T-SOD均呈NAC浓度增加而降低的趋势,并且Bmil+/--NSCs较WT-NSCs下降更明显。上述结果提示低浓度的H202或Bmil半剂量缺失导致的内源性ROS轻度上升均对NSCs增殖有促进作用,但给予外源性H202导致胞内ROS的进一步上升或给予NAC导致胞内ROS下降,对NSCs增殖及自更新能力产生的抑制作用。以上研究工作有助于深化认识以Bmil和ROS在调控神经发生中的交互作用,为治疗脑发育障碍及神经退行性疾病提供新思路。
[Abstract]:As a potential treatment for neurodegenerative diseases and the repair of central nerve damage, the proliferation of neural stem cells (NSCs) is regulated by many factors, among which reactive oxygen species (Ros) is one of them. The current results suggest that low concentrations of ROS, which do not cause oxidative stress, can be used as intracellular signal transduction molecules. Bmi1) gene is an important transcription suppressor gene in the multicomb family. P16 and P19 may play an important role in the process of NSCs proliferation and self-renewal through negative regulation. Moreover, Bmi1 could regulate mitochondrial function and then regulate ROS level to maintain intracellular redox homeostasis. Bmi1 / -) could down-regulate P16 and P19 in NSCs, but the proliferative ability of NSCs was not significantly different from that of wild-type WTS. The underlying mechanism for this phenomenon is unclear. We speculate that the down-regulation of Bmi1 leads to a slight up-regulation of ROS, which promotes the proliferation of NSCs and counteracts the blocking effect of NSCs proliferation caused by the abnormal regulation of cyclin triggered by down-regulation of Bmi1 gene. To verify this hypothesis, NSCs derived from WT and Bmi1 r-newborn mice brain was cultured in vitro and treated with different concentrations of H202 and antioxidant NAC to regulate the intracellular ROS level. The effects of ROS and Bmi-1 on the proliferation of NSCs and the mechanism of their interaction were investigated by means of intracellular ROS detection. The results showed that there was no significant difference in proliferation and self-renewal between Bmi1 / NSCs and WT-NSCs in basal state, but the intracellular ROS level increased slightly. 2) the intracellular ROS level of WT-NSCs was slightly up-regulated after 24 hours of treatment with 1 渭 M H2O2, which was consistent with this. The number of neurospheres increased slightly and the cell viability of NSCs increased significantly. The insertion ratio of BrdU was also increased, although the diameter of NSCs did not change significantly. The total antioxidant activity (T-AOC) and total superoxide dismutase activity (T-SOD) increased significantly in Bmi1 / -NSCs compared with WT-NSCs, but the other indexes were lower than those in basic state, and there was significant difference with WT-NSCs. After treated with 10 渭 M H2O2 for 24 h, the intracellular ROS of Bmil / -NSCs increased more significantly than that of WT-NSCs. The above indexes decreased in both genotypes of NSCs, but were more significant in Bmmi1 / Bmmi1 group. After treatment with 100 渭 M H2O2 for 24 h, the intracellular ROS levels of both genotypes NSCs were significantly decreased, and the other markers were further down-regulated, except that the BrdU insertion ratio of Bmil / -NSCs was lower than that of VT-NSCs. There was no significant difference between the two genotypes. 3) after being treated with different concentrations of ROS scavenger NAC for 24 h, the intracellular ROS level and the number of neurospheres in WT-NSCs were determined. The diameter and the ratio of BrdU insertion, T-AOC and T-SOD decreased with the increase of NAC concentration, and Bmil / -NSCs decreased more obviously than WT-NSCs. These results suggest that mild increase of endogenous ROS induced by low concentration of H202 or half dose of Bmil can promote the proliferation of NSCs, but exogenous H202 can induce further increase of intracellular ROS or decrease of intracellular ROS induced by NAC. Inhibition of NSCs proliferation and self-renewal ability. These studies are helpful to further understand the interaction of Bmil and ROS in the regulation of neurogenesis and provide new ideas for the treatment of brain dysplasia and neurodegenerative diseases.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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