【作者】 王宁；

【导师】 孙莉；

【作者基本信息】 天津医科大学 ， 神经病学， 2015， 硕士

【摘要】 研究目的:观察过氧化氢(H2O2)诱导的体外原代培养皮层神经元损伤中,过氧化物酶体增殖物激活受体g(PPARg)的表达及活性改变,以及PPARg激活或抑制对神经元过氧化氢损伤产生的影响,探讨PPARg对神经元氧化应激损伤的保护作用。实验方法:1、原代培养皮层神经元:取出生24h之内Sprague-Dawley新生鼠皮层神经元细胞进行体外原代培养,培养7天(d7)进行纯度鉴定,并用于各种处理和检测。2、神经元过氧化氢损伤模型:以不同浓度的过氧化氢作用于d7神经元,于不同作用时间后进行评价,选取合适浓度及作用时间应用于后续实验。3、细胞形态学观察:包括光镜下观察,即将不同组别细胞置于将倒置相差显微镜下,观察其形态变化,并采集图像;神经元MAP2免疫荧光染色,即用MAP2抗体标记神经元,荧光显微镜下观察不同组别细胞形态完整性的改变,并采集图像。4、MTT比色法检测细胞相对存活率,即检测不同组别神经元经处理后细胞活性的改变。5、分别提取总蛋白和核蛋白,Western blotting法检测不同组别细胞内PPARγ、p-PPARγ(磷酸化PPARγ)、p-ERK1/2(磷酸化ERK1/2,即ERK1/2的活性形式)、Bcl-2、活化的Caspase-3、Nrf2的表达水平。6、qRT-PCR法观察PPARγ靶基因:提取不同组别神经元RNA,qRT-PCR技术对PPARγ靶基因表达水平进行检测。7、ELISA法检测PPARγ-DNA结合活性:应用PPARγ转录因子分析试剂盒检测不同组别神经元PPARγ与过氧化物酶体增殖物反应元件(PPRE)的结合活性。8、反义寡核苷酸法降低神经元PPARγ:用PPARγ反义寡核苷酸处理原代培养d7神经元细胞,抑制PPARγ表达。实验结果:1、体外培养正常皮层神经元2d后,大多细胞呈圆形或椭圆形,可见较细小的细胞突起,至第7天时神经元胞体立体感强,形态饱满,突起变长且增粗,并相互交织成网。用神经元特异性抗体微管相关蛋白2(MAP2)对进行免疫荧光染色,鉴定神经元阳性率达95%以上,是相对纯的神经元培养,可用于后续实验。2、将过氧化氢作用于神经元,可造成神经元损伤,轻者细胞肿胀变圆,细胞突起断裂或消失;重者细胞发生崩解,可见较多细胞碎片,并呈现剂量-时程依赖效应。选取浓度为500mM、作用6~24小时进行后续实验。3、过氧化氢抑制神经元PPARg蛋白表达,同时增加PPARg灭活;抑制ERK1/2激活可以拮抗过氧化氢对PPARg的灭活作用。4、外源性给予PPARg激动剂罗格列酮减轻神经元过氧化氢损伤、增加Bcl-2及Nrf2表达,同时抑制活化的Caspase-3表达;而PPARg抑制剂GW9662可以拮抗罗格列酮的保护作用、降低Bcl-2及Nrf2表达、增加活化的Caspase-3表达。5、降低PPARg表达,对正常基础状态下的神经元形态和存活率无明显影响,但使神经元对过氧化氢损伤更敏感,增加过氧化氢诱导的Bcl-2、Nrf2、活化的Caspase-3表达的改变,同时可以降低外源性给予PPARg激动剂罗格列酮所产生的保护作用。实验结论:1、在体外原代培养皮层神经元中,过氧化氢可能通过负性调节PPARg(即表达下调和活性降低)引起神经元损伤。2、在体外原代培养皮层神经元中,外源性激活PPARγ可以保护神经元过氧化氢损伤,而神经元自身PPARγ的表达可能是神经元自我保护机制之一。3、PPARγ的神经保护作用可能部分通过抑制活化的Caspase-3、上调Bcl-2抗凋亡蛋白,以及增加抗氧化应激转录因子Nrf2的表达实现的。更多还原

【Abstract】 ObjectiveTo observe the changes of PPARγ expression and activity induced by hydrogen peroxide(H2O2) in primary cultured cortical neurons, as well as the protective effects of neuronal PPARγ to H2O2-induced cellular injury. Methods1. Primary culture of cortical neurons: Cortical neurons from new born SD rats(within 24h) were cultured in vitro for 7 days. After the purity identification, neurons are used in the following experiments.2. H2O2-induced neuronal injury: H2O2 was added to neurons to elicit oxidative celluar insult. Neurons of d7 was exposed to H2O2 of different concentrations for 24 h or H2O2 of the indicated concentration for different times. Appropriate concentration and time course was selected for the following experiments.3. Morphological observation of neurons:The morphological damage was examined with an inverted phase contrast microscope. Immunofluorescent staining of MAP2 was also used to observe the gross morphological features of neurons.4. MTT assay: MTT assay was used to determine the cell viability after H2O2 exposure.5. Western blot analysis: Western blotting was chosen to observe the expressions of PPARγ, p-PPARγ, ERK1/2, p-ERK1/2, Bcl-2, cleaved caspase-3, and Nrf2 in neurons with different treatments.6. RT-PCR analysis:RT-PCR was used to detect mRNA expression of target genes of PPARγ in neurons with different treatments.7. ELISA assay: ELISA assay was applied to explore PPARγ-DNA binding activity in neurons with different treatments.8. The antisense oligonucleotides approach(asON): an asON-based strategy was used to downregulate PPARγ expression in neurons to assess the protective potential of neuronal PPARγ towards H2O2-induced injury.Results1. The primary cortical neurons were adhered to the dishes with a round or elliptical body and tiny neuritis when cultured for the first 2 days. When cultured for 7 days, the neurons were of plump size with plenty of neuritis interweaving to form a net. And the purity of neurons was up to 95% by immunofluorescent staining of MAP2, capable of being used in the follow-up experiments.2. Hydrogen peroxide can cause neuron damage in a concentration and time dependent manner: Cells lost the polygonal shape, some cells appeared swelling and others shrunken with surface blebbing. There was general loss of neurites, which were either irregular or fragmented. Hydrogen peroxide in the concentration of 500mM was selected as the injured model for the following observation.3. Hydrogen peroxide suppressed PPARγ protein level of neurons, and at the same time increased PPARγ inactivation. ERK1/2 activation inhibitor can block hydrogen peroxide-induced PPARγ inactivation.4. PPARγ activator Rosiglitazone(Ros) protected neurons against hydrogen peroxide injury, inhibited hydrogen peroxide-induced decrease in Bcl-2, Nrf2 expression and the increase in cleaved caspase-3 level. Furthermore, the above effects of Ros can be reversed by the PPARγ inhibitor GW9662.5. Neurons with PPARγ downregulation by antisense approach(asON) showed more damage to hydrogen peroxide and the enhancement in hydrogen peroxide-induced alterations in Bcl-2, Nrf2, and cleaved caspase-3 expression. Also, PPARγ agonist Ros based neuroprotective effect was reduced. Conclusion1. In primary cultured cortical neurons, hydrogen peroxide may induce cell injury by negative regulation of PPARγ.2. In primary cultured cortical neurons, PPARγ can protect neurons from hydrogen peroxide injury. The intrinsic PPARγ expression in neurons may be one of self-protective defense of neurons.3. PPARγ may exert its neuroprotective effects partly through upregulation of Bcl-2 and Nrf2, as well as downregulation of caspase-3.更多还原