mfat-1基因对氯化钴诱导的成体神经干细胞缺氧性损伤保护作用的研究
发布时间：2019-05-21 08:33
【摘要】：研究背景:随着人们生活方式和饮食习惯的改变,缺血性脑卒中已经成为成年人致死致残以及神经功能损伤的主要原因之一。据统计缺血性脑卒中占脑卒中的比例已超过80%,然而现在临床仍然缺乏有效的治疗手段。成体神经干细胞替代性治疗方法在神经系统结构和功能恢复治疗方面将有广阔的应用前景。但是,成体神经干细胞替代疗法应用于缺血性脑卒中临床治疗的最大障碍是在缺血半暗带区缺血缺氧的微环境对成体神经干细胞活性和功能的损伤。所以,能否找到一种保护缺血半暗带区成体神经干细胞免受缺血缺氧性损伤的方法是成体神经干细胞替代疗法临床应用的关键。目的:通过建立成体神经干细胞体外缺血缺氧性损伤模型,探讨mfat-1基因是否对氯化钴诱导的成体神经干细胞缺氧性损伤具有保护作用,同时进一步研究其内在机制,为成体神经干细胞替代疗法的临床应用打下基础。方法:mfat-1转基因小鼠和其同窝阴性小鼠饲养至8-12周龄,基因型鉴定完成后,无菌环境下提取两组小鼠大脑Subventricular xone(SVZ)区神经干细胞,培养至第三代用于以下实验。细胞免疫组化鉴定神经干细胞标志蛋白Nestin,通过气相色谱技术分别测定mfat-1组及同窝阴性组鼠脑和神经干细胞n-3/n-6的比值。通过氯化钴诱导体外构建成体神经干细胞缺血缺氧模型;通过测定两组细胞的细胞活性,细胞凋亡率和细胞增殖率来研究mfat-1基因对成体神经干细胞体外缺氧性损伤的保护作用。为了进一步探究mfat-1基因的作用机制,我们分别测定了两组细胞活性氧水平以及谷胱甘肽表达水平,确定了mfat-1基因的保护机制是通过促进抗氧化应激损伤来完成的。我们选取了抗氧化应激的关键信号通路Nrf2/ARE信号通路,分别检测了Nrf2及下游基因HO-1、NQO-1、GCLC mRNA表达水平以及蛋白表达水平。结果:mfat-1基因可以增强氯化钴诱导的缺氧性损伤中成体神经干细胞的细胞活性,与此同时抑制氯化钻介导的成体神经干细胞的凋亡。通过BrdU标记实验表明,在氯化钻诱导的体外缺氧损伤模型中,mfat-1组的细胞增殖率明显高于同窝阴性对照组。活性氧水平以及细胞谷胱甘肽表达水平检测结果显示mfat-1组细胞活性氧水平明显低于同窝阴性对照组,同时谷肮甘肽表达量明显高于同窝阴性对照组。实时定量PCR检测结果显示Nrf2及其下游基因的mRNA的表达量均高于同窝阴性对照组。Western blot检测结果显示Nrf2及其下游基因HO-1、NQO-1、GCLC的蛋白表达量均高于对照组。结论:mfat-1基因对氯化钴介导的成体神经干细胞缺氧性损伤具有保护作用,其潜在的机制可能是激活Nrf2/ARE信号通路,来上调抗氧化因子及二相解毒酶的表达。这为成体神经干细胞替代疗法的临床应用奠定了理论基础。
[Abstract]:Background: with the change of people's lifestyle and eating habits, ischemic stroke has become one of the main causes of death, disability and neurological impairment in adults. According to statistics, ischemic stroke accounts for more than 80% of stroke, but there is still a lack of effective treatment. Alternative therapy of adult neural stem cells will have a broad application prospect in the treatment of structural and functional recovery of nervous system. However, the biggest obstacle to the clinical treatment of ischemic stroke by adult neural stem cell replacement therapy is the damage to the activity and function of adult neural stem cells in the ischemic and anoxic microenvironment in the ischemic penumbra. Therefore, whether we can find a way to protect adult neural stem cells from ischemic and anoxic injury in ischemic penumbra is the key to the clinical application of adult neural stem cell replacement therapy. Objective: to establish a model of ischemic and anoxic injury of adult neural stem cells in vitro, and to explore whether mfat-1 gene has protective effect on anoxic injury of adult neural stem cells induced by cobalt chloride, and to further study its internal mechanism. It lays a foundation for the clinical application of adult neural stem cell replacement therapy. Methods: mfat-1 transgenic mice and their neonate negative mice were raised to 8 to 12 weeks of age. After genotypic identification, neural stem cells from Subventricular xone (SVZ) region of brain of two groups of mice were extracted in aseptic environment and cultured to the third generation for the following experiments. The ratio of n-3/n-6 in brain and neural stem cells of mfat-1 group and identical fossa negative group was determined by gas chromatography (GC). The model of ischemia and hypoxia of adult neural stem cells was established by induction of cobalt chloride in vitro. The protective effect of mfat-1 gene on hypoxia injury of adult neural stem cells in vitro was studied by measuring the cell activity, apoptosis rate and cell proliferation rate of the two groups of cells. In order to further explore the mechanism of mfat-1 gene, we measured the level of reactive oxygen species (Ros) and the expression of glutathione in two groups of cells, and determined that the protective mechanism of mfat-1 gene was achieved by promoting antioxidant stress injury. We selected the Nrf2/ARE signaling pathway, which is the key signaling pathway of antioxidant stress, and detected the HO-1,NQO-1,GCLC mRNA expression level and protein expression level of Nrf2 and downstream genes, respectively. Results: mfat-1 gene could enhance the cell activity of adult neural stem cells in anoxic injury induced by cobalt chloride, and inhibit the apoptosis of adult neural stem cells mediated by chloride drill at the same time. BrdU labeling test showed that the cell proliferation rate of mfat-1 group was significantly higher than that of the control group induced by chloride drill in vitro. The results showed that the level of reactive oxygen species (Ros) in mfat-1 group was significantly lower than that in the negative control group, and the expression of glutathion was significantly higher than that in the negative control group. The results of real-time quantitative PCR showed that the expression of mRNA in Nrf2 and its downstream genes was higher than that in the control group. Western blot showed that the protein expression of Nrf2 and its downstream gene HO-1,NQO-1,GCLC was higher than that in the control group. Conclusion: mfat-1 gene has protective effect on hypoxia injury of adult neural stem cells mediated by cobalt chloride, and its potential mechanism may be to activate Nrf2/ARE signaling pathway to up-regulate the expression of antioxidant factor and two-phase detoxification enzyme. This lays a theoretical foundation for the clinical application of adult neural stem cell replacement therapy.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
本文编号：2481969
[Abstract]:Background: with the change of people's lifestyle and eating habits, ischemic stroke has become one of the main causes of death, disability and neurological impairment in adults. According to statistics, ischemic stroke accounts for more than 80% of stroke, but there is still a lack of effective treatment. Alternative therapy of adult neural stem cells will have a broad application prospect in the treatment of structural and functional recovery of nervous system. However, the biggest obstacle to the clinical treatment of ischemic stroke by adult neural stem cell replacement therapy is the damage to the activity and function of adult neural stem cells in the ischemic and anoxic microenvironment in the ischemic penumbra. Therefore, whether we can find a way to protect adult neural stem cells from ischemic and anoxic injury in ischemic penumbra is the key to the clinical application of adult neural stem cell replacement therapy. Objective: to establish a model of ischemic and anoxic injury of adult neural stem cells in vitro, and to explore whether mfat-1 gene has protective effect on anoxic injury of adult neural stem cells induced by cobalt chloride, and to further study its internal mechanism. It lays a foundation for the clinical application of adult neural stem cell replacement therapy. Methods: mfat-1 transgenic mice and their neonate negative mice were raised to 8 to 12 weeks of age. After genotypic identification, neural stem cells from Subventricular xone (SVZ) region of brain of two groups of mice were extracted in aseptic environment and cultured to the third generation for the following experiments. The ratio of n-3/n-6 in brain and neural stem cells of mfat-1 group and identical fossa negative group was determined by gas chromatography (GC). The model of ischemia and hypoxia of adult neural stem cells was established by induction of cobalt chloride in vitro. The protective effect of mfat-1 gene on hypoxia injury of adult neural stem cells in vitro was studied by measuring the cell activity, apoptosis rate and cell proliferation rate of the two groups of cells. In order to further explore the mechanism of mfat-1 gene, we measured the level of reactive oxygen species (Ros) and the expression of glutathione in two groups of cells, and determined that the protective mechanism of mfat-1 gene was achieved by promoting antioxidant stress injury. We selected the Nrf2/ARE signaling pathway, which is the key signaling pathway of antioxidant stress, and detected the HO-1,NQO-1,GCLC mRNA expression level and protein expression level of Nrf2 and downstream genes, respectively. Results: mfat-1 gene could enhance the cell activity of adult neural stem cells in anoxic injury induced by cobalt chloride, and inhibit the apoptosis of adult neural stem cells mediated by chloride drill at the same time. BrdU labeling test showed that the cell proliferation rate of mfat-1 group was significantly higher than that of the control group induced by chloride drill in vitro. The results showed that the level of reactive oxygen species (Ros) in mfat-1 group was significantly lower than that in the negative control group, and the expression of glutathion was significantly higher than that in the negative control group. The results of real-time quantitative PCR showed that the expression of mRNA in Nrf2 and its downstream genes was higher than that in the control group. Western blot showed that the protein expression of Nrf2 and its downstream gene HO-1,NQO-1,GCLC was higher than that in the control group. Conclusion: mfat-1 gene has protective effect on hypoxia injury of adult neural stem cells mediated by cobalt chloride, and its potential mechanism may be to activate Nrf2/ARE signaling pathway to up-regulate the expression of antioxidant factor and two-phase detoxification enzyme. This lays a theoretical foundation for the clinical application of adult neural stem cell replacement therapy.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
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