【摘要】：研究背景:隨著人們生活方式和飲食習(xí)慣的改變,缺血性腦卒中已經(jīng)成為成年人致死致殘以及神經(jīng)功能損傷的主要原因之一。據(jù)統(tǒng)計(jì)缺血性腦卒中占腦卒中的比例已超過80%,然而現(xiàn)在臨床仍然缺乏有效的治療手段。成體神經(jīng)干細(xì)胞替代性治療方法在神經(jīng)系統(tǒng)結(jié)構(gòu)和功能恢復(fù)治療方面將有廣闊的應(yīng)用前景。但是,成體神經(jīng)干細(xì)胞替代療法應(yīng)用于缺血性腦卒中臨床治療的最大障礙是在缺血半暗帶區(qū)缺血缺氧的微環(huán)境對(duì)成體神經(jīng)干細(xì)胞活性和功能的損傷。所以,能否找到一種保護(hù)缺血半暗帶區(qū)成體神經(jīng)干細(xì)胞免受缺血缺氧性損傷的方法是成體神經(jīng)干細(xì)胞替代療法臨床應(yīng)用的關(guān)鍵。目的:通過建立成體神經(jīng)干細(xì)胞體外缺血缺氧性損傷模型,探討mfat-1基因是否對(duì)氯化鈷誘導(dǎo)的成體神經(jīng)干細(xì)胞缺氧性損傷具有保護(hù)作用,同時(shí)進(jìn)一步研究其內(nèi)在機(jī)制,為成體神經(jīng)干細(xì)胞替代療法的臨床應(yīng)用打下基礎(chǔ)。方法:mfat-1轉(zhuǎn)基因小鼠和其同窩陰性小鼠飼養(yǎng)至8-12周齡,基因型鑒定完成后,無菌環(huán)境下提取兩組小鼠大腦Subventricular xone(SVZ)區(qū)神經(jīng)干細(xì)胞,培養(yǎng)至第三代用于以下實(shí)驗(yàn)。細(xì)胞免疫組化鑒定神經(jīng)干細(xì)胞標(biāo)志蛋白Nestin,通過氣相色譜技術(shù)分別測(cè)定mfat-1組及同窩陰性組鼠腦和神經(jīng)干細(xì)胞n-3/n-6的比值。通過氯化鈷誘導(dǎo)體外構(gòu)建成體神經(jīng)干細(xì)胞缺血缺氧模型;通過測(cè)定兩組細(xì)胞的細(xì)胞活性,細(xì)胞凋亡率和細(xì)胞增殖率來研究mfat-1基因?qū)Τ审w神經(jīng)干細(xì)胞體外缺氧性損傷的保護(hù)作用。為了進(jìn)一步探究mfat-1基因的作用機(jī)制,我們分別測(cè)定了兩組細(xì)胞活性氧水平以及谷胱甘肽表達(dá)水平,確定了mfat-1基因的保護(hù)機(jī)制是通過促進(jìn)抗氧化應(yīng)激損傷來完成的。我們選取了抗氧化應(yīng)激的關(guān)鍵信號(hào)通路Nrf2/ARE信號(hào)通路,分別檢測(cè)了Nrf2及下游基因HO-1、NQO-1、GCLC mRNA表達(dá)水平以及蛋白表達(dá)水平。結(jié)果:mfat-1基因可以增強(qiáng)氯化鈷誘導(dǎo)的缺氧性損傷中成體神經(jīng)干細(xì)胞的細(xì)胞活性,與此同時(shí)抑制氯化鉆介導(dǎo)的成體神經(jīng)干細(xì)胞的凋亡。通過BrdU標(biāo)記實(shí)驗(yàn)表明,在氯化鉆誘導(dǎo)的體外缺氧損傷模型中,mfat-1組的細(xì)胞增殖率明顯高于同窩陰性對(duì)照組�；钚匝跛揭约凹�(xì)胞谷胱甘肽表達(dá)水平檢測(cè)結(jié)果顯示mfat-1組細(xì)胞活性氧水平明顯低于同窩陰性對(duì)照組,同時(shí)谷骯甘肽表達(dá)量明顯高于同窩陰性對(duì)照組。實(shí)時(shí)定量PCR檢測(cè)結(jié)果顯示Nrf2及其下游基因的mRNA的表達(dá)量均高于同窩陰性對(duì)照組。Western blot檢測(cè)結(jié)果顯示Nrf2及其下游基因HO-1、NQO-1、GCLC的蛋白表達(dá)量均高于對(duì)照組。結(jié)論:mfat-1基因?qū)β然捊閷?dǎo)的成體神經(jīng)干細(xì)胞缺氧性損傷具有保護(hù)作用,其潛在的機(jī)制可能是激活Nrf2/ARE信號(hào)通路,來上調(diào)抗氧化因子及二相解毒酶的表達(dá)。這為成體神經(jīng)干細(xì)胞替代療法的臨床應(yīng)用奠定了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743.3

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