氧—糖剥夺致皮层神经元一氧化氮的变化及对caspase-3的影响
发布时间：2019-05-31 17:51
【摘要】：目的：观察体外培养皮层神经元细胞进行糖-氧剥夺处理和L-NAME干预后培养基中一氧化氮（NO）含量的变化，以及对神经元细胞caspase-3的影响。 方法：取新生大鼠（24h）大脑皮层组织，消化后种植在有多聚-L-赖氨酸包被的六孔培养皿中，以含10%胎牛血清DEME-HG液种植，4-8小时后换用含B27的Neurobasal培养基维持饲养。于不同时间在倒置相差显微镜下观察形态变化，分别采用免疫组化对神经元特异性标记物NSE进行鉴定。培养至第7天时，选取生长良好、均一的神经元细胞随机分为3组。分别为：正常对照组，氧-糖剥夺建立细胞损伤模型组，细胞损伤模型+L-N-硝基精氨酸甲脂。采用硝酸还原法检测各组培养基中NO的含量，Western-blot检测caspase-3、NSE蛋白表达，半定量RT-PCR检测caspase-3mRNA表达。 结果：1.成功原代培养新生大鼠皮层神经元，经NSE免疫组化鉴定神经元纯度达90%以上。 2.氧-糖剥夺致神经元损伤组培养基中NO含量(1.77±0.17)umoL/L，与正常对照组培养基中NO的含量(0.93±0．15)umoL/L和细胞损伤模型+L-N-硝基精氨酸甲脂组培养基中含量(0.99±0.10)umol／L比较差异有统计学意义(p0.05)；正常对照组培养基中NO的含量与细胞损伤模型+L-N-硝基精氨酸甲脂组培养基中含量比较，差异无统计学意义(p0.05)。 3.氧-糖剥夺致神经元损伤组中神经元的caspase-3蛋白表达和mRNA明显高于正常对照组和细胞损伤模型+L-N-硝基精氨酸甲脂组中的神经元的caspase-3蛋白表达和mRNA(p0.05)；正常对照组中caspase-3蛋白表达和mRNA表达与细胞损伤模型+L-N-硝基精氨酸甲脂组中caspase-3蛋白表达和mRNA表达比较，差异无统计学意义(p0.05)。 4.氧-糖剥夺致神经元损伤组中神经元的NSE蛋白表达明显低于正常对照组和细胞损伤模型+L-N-硝基精氨酸甲脂组中的神经元的NSE表达(p0.05)。正常对照组中NSE蛋白表达与细胞损伤模型+L-N-硝基精氨酸甲脂组中NSE蛋白表达比较，差异无统计学意义(p0.05)。 结论：1.采用无血清原代培养新生SD大鼠皮层神经元细胞，差速贴壁进行纯化，本方法简单易行神经元纯度较高，可作为神经元体外培养的良好模型用于以后的研究。 2.氧-糖剥夺可导致体外培养皮层神经元细胞一氧化氮（NO）产生过量，并造成神经元的损伤。 3.合成NO的关键酶NOS的抑制剂L-N-硝基精氨酸甲脂（L-NAME）能抑制NOS活性，，减少NO的产生，降低caspase-3表达，从而减弱氧-糖剥夺致皮层神经元的损伤作用，起到神经保护作用。
[Abstract]:Aim: to observe the changes of nitric oxide (no) (NO) content in culture medium after glucose-oxygen deprivation and L-NAME intervention in vitro and its effect on caspase-3 of neuron cells. Methods: the cerebral cortex of neonatal rats (24 h) was digested and planted in a six-hole petri dish coated with polyL-lysine and planted with 10% fetal bovine serum DEME-HG solution. After 8 hours, Neurobasal medium containing B27 was used to maintain feeding. The morphological changes were observed under inverted phase contrast microscope at different time, and the neuron specific marker NSE was identified by immunohistochemistry. On the 7th day, the well-growing and homogeneous neurons were randomly divided into three groups. In the normal control group, the cell injury model group was established by oxygen-glucose deprivation, and the cell injury model L 鈮
本文编号：2489906
[Abstract]:Aim: to observe the changes of nitric oxide (no) (NO) content in culture medium after glucose-oxygen deprivation and L-NAME intervention in vitro and its effect on caspase-3 of neuron cells. Methods: the cerebral cortex of neonatal rats (24 h) was digested and planted in a six-hole petri dish coated with polyL-lysine and planted with 10% fetal bovine serum DEME-HG solution. After 8 hours, Neurobasal medium containing B27 was used to maintain feeding. The morphological changes were observed under inverted phase contrast microscope at different time, and the neuron specific marker NSE was identified by immunohistochemistry. On the 7th day, the well-growing and homogeneous neurons were randomly divided into three groups. In the normal control group, the cell injury model group was established by oxygen-glucose deprivation, and the cell injury model L 鈮
本文编号：2489906
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