铅致原代培养大鼠皮质神经细胞氧化应激损伤及JWA mRNA表达改变
发布时间：2018-10-14 09:00
【摘要】：铅是一种化学性质稳定的重金属毒物,神经系统是铅毒作用的重要靶器官。铅暴露可降低细胞中超氧化物歧化酶、过氧化氢酶等抗氧化酶的活性及谷胱甘肽等抗氧化物质的含量,引起细胞氧化损伤,因此铅的神经毒性与氧化应激及其所致氧化损伤密切相关。JWA是活跃的环境应答基因,广泛参与调控多种环境因素诱导的氧化应激,保护细胞免受氧化损伤。目前尚未见JWA在铅毒性中作用的相关报道,本研究以体外培养的新生大鼠皮质神经细胞为研究对象,研究JWA在铅神经毒性中的表达变化及可能机制。目的:观察铅对大鼠大脑皮质神经细胞中各氧化应激指标以及JWA基因表达的影响,探讨JWA基因参与神经细胞铅氧化应激作用可能机制,为进一步从分子水平探讨铅的神经毒性机制提供新的线索和依据。方法:取新生24 h内的SD大鼠皮质神经细胞进行原代培养并用免疫细胞化学方法鉴定;将体外生长良好的皮质神经细胞,分别用终浓度为0μmol/L、31μmol/L、54μmol/L、92μmol/L、150μmol/L醋酸铅神经细胞维持培养液处理一定时间,MTT法检测细胞存活情况,并检测细胞超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、过氧化氢酶(CAT)活力和谷胱甘肽(GSH)含量,QRT-PCR技术检测皮质神经细胞JWA mRNA的表达量。结果:(1)形态学观察和免疫组织化学鉴定发现,体外原代培养的皮质神经细胞在培养第7 d左右生长状态良好,神经细胞所占比例较高,可用于之后铅损伤模型的建立。(2)随着醋酸铅浓度的逐渐升高,培养的大鼠皮质神经细胞数量逐渐减少。染铅24 h和48 h,92μmol/L和150μmol/L醋酸铅组同其他剂量组间差异有统计学意义(P0.05),92μmol/L醋酸铅作用下平均OD值分别为0.218±0.008和0.181±0.006,而150μmol/L醋酸铅作用下平均OD值分别为0.199±0.008和0.166±0.009。筛选出醋酸铅的适宜作用浓度为92μmol/L,适宜作用时间为24 h。(3)不同浓度醋酸铅分别作用皮质神经元6 h、12 h、24h、48 h,SOD活力、CAT活力和GSH含量较对照组均有明显下降(P0.05),而MDA含量较对照组均有明显升高(P0.05),皮质神经细胞内SOD活力、CAT活力和GSH含量与染铅剂量呈负相关关系(P0.01),MDA含量与染铅剂量呈正相关关系(P0.01)。(4)随着铅含量的升高,各铅处理组细胞JWA mRNA的表达水平呈逐渐下降趋势(P0.05);铅处理的同时加入适当的超氧化物歧化酶,JWA mRNA的表达量差异无统计学意义(P0.05),而铅处理同时加入适量的过氧化氢酶,JWA mRNA的表达量升高,且差异有统计学意义(P0.05);JWA mRNA表达量与SOD活力、CAT活力和GSH含量呈正相关关系,与MDA含量呈负相关关系(P0.01)。结论:铅暴露可导致原代培养的大鼠皮质神经细胞发生氧化应激损伤并影响基因JWA的表达。
[Abstract]:Lead is a kind of heavy metal poison with stable chemical properties. The nervous system is an important target organ of lead toxicity. Lead exposure can reduce the activities of superoxide dismutase, catalase and other antioxidant enzymes, and the content of antioxidants such as glutathione, thus causing oxidative damage to cells. Therefore, the neurotoxicity of lead is closely related to oxidative stress and oxidative damage. JWA is an active environmental response gene, which is widely involved in regulating oxidative stress induced by many environmental factors and protecting cells from oxidative damage. There is no report on the role of JWA in lead toxicity. In this study, the expression of JWA in lead neurotoxicity and its possible mechanism were studied in cultured neonatal rat cortical nerve cells. Aim: to observe the effects of lead on the oxidative stress indexes and the expression of JWA gene in rat cerebral cortical neurons, and to explore the possible mechanism of JWA gene involved in the oxidative stress of nerve cells. It provides a new clue and basis for further exploring the neurotoxic mechanism of lead at molecular level. Methods: the cortical neurons of SD rats within 24 hours were cultured and identified by immunocytochemistry. The cells were treated with 0 渭 mol/L,31 渭 mol/L,54 渭 mol/L,92 渭 mol/L,150 渭 mol/L lead acetate culture medium for a certain time, the survival of the cells was detected by MTT method, the (SOD) activity of superoxide dismutase (SOD) and the content of MDA (MDA) were measured. The activity of catalase (CAT) and the content of glutathione (GSH) were measured. The expression of JWA mRNA in cortical neurons was detected by QRT-PCR. Results: (1) morphological observation and immunohistochemical analysis showed that the primary cultured cortical neurons grew well on the 7th day of culture, and the proportion of neurons was high. It can be used to establish the lead injury model. (2) with the increasing of lead acetate concentration, the number of cultured cortical nerve cells decreased gradually. There was significant difference between lead exposure group and other dose groups in lead exposure for 24 h and 48 h (P0.05). The average OD values of lead acetate group exposed to lead acetate at 92 渭 mol/L were 0.218 卤0.008 and 0.181 卤0.006, respectively, and the average OD values of lead acetate group exposed to 150 渭 mol/L lead acetate were 0.199 卤0.008 and 0.166 卤0.009, respectively. The optimum concentration of lead acetate was 92 渭 mol/L, for 24 h. (3) the SOD activity, CAT activity and GSH content of cortical neurons treated with lead acetate for 6 h, 12 h, 24 h and 48 h were significantly lower than those of the control group (P0.05), while the content of MDA was significantly lower than that of the control group (P0.05). SOD activity, CAT activity and GSH content in cortical nerve cells were negatively correlated with lead exposure dose (P0.01). (4). The expression of JWA mRNA in the cells of each lead treatment group showed a decreasing trend (P0.05), while the expression of superoxide dismutase, JWA mRNA was not significantly different (P0.05), while the lead treatment with appropriate amount of hydrogen peroxide added at the same time (P0.05). The expression of enzyme, JWA mRNA was increased, The difference was statistically significant (P0.05) the expression of); JWA mRNA was positively correlated with SOD activity, CAT activity and GSH content, and negatively correlated with MDA content (P0.01). Conclusion: lead exposure can induce oxidative stress injury and affect gene JWA expression in primary cultured rat cortical neurons.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2269996
[Abstract]:Lead is a kind of heavy metal poison with stable chemical properties. The nervous system is an important target organ of lead toxicity. Lead exposure can reduce the activities of superoxide dismutase, catalase and other antioxidant enzymes, and the content of antioxidants such as glutathione, thus causing oxidative damage to cells. Therefore, the neurotoxicity of lead is closely related to oxidative stress and oxidative damage. JWA is an active environmental response gene, which is widely involved in regulating oxidative stress induced by many environmental factors and protecting cells from oxidative damage. There is no report on the role of JWA in lead toxicity. In this study, the expression of JWA in lead neurotoxicity and its possible mechanism were studied in cultured neonatal rat cortical nerve cells. Aim: to observe the effects of lead on the oxidative stress indexes and the expression of JWA gene in rat cerebral cortical neurons, and to explore the possible mechanism of JWA gene involved in the oxidative stress of nerve cells. It provides a new clue and basis for further exploring the neurotoxic mechanism of lead at molecular level. Methods: the cortical neurons of SD rats within 24 hours were cultured and identified by immunocytochemistry. The cells were treated with 0 渭 mol/L,31 渭 mol/L,54 渭 mol/L,92 渭 mol/L,150 渭 mol/L lead acetate culture medium for a certain time, the survival of the cells was detected by MTT method, the (SOD) activity of superoxide dismutase (SOD) and the content of MDA (MDA) were measured. The activity of catalase (CAT) and the content of glutathione (GSH) were measured. The expression of JWA mRNA in cortical neurons was detected by QRT-PCR. Results: (1) morphological observation and immunohistochemical analysis showed that the primary cultured cortical neurons grew well on the 7th day of culture, and the proportion of neurons was high. It can be used to establish the lead injury model. (2) with the increasing of lead acetate concentration, the number of cultured cortical nerve cells decreased gradually. There was significant difference between lead exposure group and other dose groups in lead exposure for 24 h and 48 h (P0.05). The average OD values of lead acetate group exposed to lead acetate at 92 渭 mol/L were 0.218 卤0.008 and 0.181 卤0.006, respectively, and the average OD values of lead acetate group exposed to 150 渭 mol/L lead acetate were 0.199 卤0.008 and 0.166 卤0.009, respectively. The optimum concentration of lead acetate was 92 渭 mol/L, for 24 h. (3) the SOD activity, CAT activity and GSH content of cortical neurons treated with lead acetate for 6 h, 12 h, 24 h and 48 h were significantly lower than those of the control group (P0.05), while the content of MDA was significantly lower than that of the control group (P0.05). SOD activity, CAT activity and GSH content in cortical nerve cells were negatively correlated with lead exposure dose (P0.01). (4). The expression of JWA mRNA in the cells of each lead treatment group showed a decreasing trend (P0.05), while the expression of superoxide dismutase, JWA mRNA was not significantly different (P0.05), while the lead treatment with appropriate amount of hydrogen peroxide added at the same time (P0.05). The expression of enzyme, JWA mRNA was increased, The difference was statistically significant (P0.05) the expression of); JWA mRNA was positively correlated with SOD activity, CAT activity and GSH content, and negatively correlated with MDA content (P0.01). Conclusion: lead exposure can induce oxidative stress injury and affect gene JWA expression in primary cultured rat cortical neurons.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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