發(fā)布時間：2019-05-21 08:33

【摘要】：研究背景:隨著人們生活方式和飲食習慣的改變,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)干細胞替代性治療方法在神經(jīng)系統(tǒng)結(jié)構(gòu)和功能恢復治療方面將有廣闊的應(yīng)用前景。但是,成體神經(jīng)干細胞替代療法應(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)境對成體神經(jīng)干細胞活性和功能的損傷。所以,能否找到一種保護缺血半暗帶區(qū)成體神經(jīng)干細胞免受缺血缺氧性損傷的方法是成體神經(jīng)干細胞替代療法臨床應(yīng)用的關(guān)鍵。目的:通過建立成體神經(jīng)干細胞體外缺血缺氧性損傷模型,探討mfat-1基因是否對氯化鈷誘導的成體神經(jīng)干細胞缺氧性損傷具有保護作用,同時進一步研究其內(nèi)在機制,為成體神經(jīng)干細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)干細胞,培養(yǎng)至第三代用于以下實驗。細胞免疫組化鑒定神經(jīng)干細胞標志蛋白Nestin,通過氣相色譜技術(shù)分別測定mfat-1組及同窩陰性組鼠腦和神經(jīng)干細胞n-3/n-6的比值。通過氯化鈷誘導體外構(gòu)建成體神經(jīng)干細胞缺血缺氧模型;通過測定兩組細胞的細胞活性,細胞凋亡率和細胞增殖率來研究mfat-1基因?qū)Τ审w神經(jīng)干細胞體外缺氧性損傷的保護作用。為了進一步探究mfat-1基因的作用機制,我們分別測定了兩組細胞活性氧水平以及谷胱甘肽表達水平,確定了mfat-1基因的保護機制是通過促進抗氧化應(yīng)激損傷來完成的。我們選取了抗氧化應(yīng)激的關(guān)鍵信號通路Nrf2/ARE信號通路,分別檢測了Nrf2及下游基因HO-1、NQO-1、GCLC mRNA表達水平以及蛋白表達水平。結(jié)果:mfat-1基因可以增強氯化鈷誘導的缺氧性損傷中成體神經(jīng)干細胞的細胞活性,與此同時抑制氯化鉆介導的成體神經(jīng)干細胞的凋亡。通過BrdU標記實驗表明,在氯化鉆誘導的體外缺氧損傷模型中,mfat-1組的細胞增殖率明顯高于同窩陰性對照組�；钚匝跛揭约凹毎入赘孰谋磉_水平檢測結(jié)果顯示mfat-1組細胞活性氧水平明顯低于同窩陰性對照組,同時谷骯甘肽表達量明顯高于同窩陰性對照組。實時定量PCR檢測結(jié)果顯示Nrf2及其下游基因的mRNA的表達量均高于同窩陰性對照組。Western blot檢測結(jié)果顯示Nrf2及其下游基因HO-1、NQO-1、GCLC的蛋白表達量均高于對照組。結(jié)論:mfat-1基因?qū)β然捊閷У某审w神經(jīng)干細胞缺氧性損傷具有保護作用,其潛在的機制可能是激活Nrf2/ARE信號通路,來上調(diào)抗氧化因子及二相解毒酶的表達。這為成體神經(jīng)干細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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.3

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