NMDA经p38MAPK信号通路诱导的皮层神经元损伤及MK801及SB203580的保护机制

发布时间：2018-07-07 09:43

本文选题：p38丝裂原活化蛋白激酶 + N-甲基-D-天冬氨酸　；参考：《辽宁医学院》2014年硕士论文

【摘要】：目的观察N-甲基-D-天门冬氨酸（NMDA）对原代培养的皮层神经元损伤的影响，应用NMDA受体（NMDAR）抑制剂MK801及p38丝裂原活化蛋白激酶（p38MAPK）抑制剂SB203580进行干预，从信号转导通路和凋亡因子两方面来探讨p38MAPK信号通路在神经元损伤中的作用和机制。方法选择NMDAR特异性激动剂NMDA复制体外培养的神经元损伤模型。培养7d的新生SD大鼠皮层神经元，随机分为5组：对照组、NMDA损伤组（NMDA50μmol/L）、MK801干预组（NMDA50μmol/L+MK80110μmol/L）、SB203580干预组（NMDA50μmol/L+SB20358010μmol/L）和联合干预组（NMDA50μmol/L+MK801和SB203580各10μmol/L）。采用抗神经元特异性稀醇化酶(NSE)免疫荧光化学染色鉴定神经元，噻唑蓝（MTT）比色实验评价细胞生存力，乳酸脱氢酶(LDH)释放率实验测定细胞损伤程度，吖啶橙/溴化乙锭(AO/EB)双重荧光染色观察细胞凋亡形态和数目，免疫细胞化学染色法（IHC）和Western blot法检测大鼠皮层神经元各组p-p38MAPK，BCL-2和BAX表达情况。结果同对照组比较，NMDA损伤组神经细胞细胞生存力显著降低，培养上清液中LDH含量明显增加，凋亡细胞显著增多，p-p38MAPK、BAX蛋白表达明显增加，而BCL-2蛋白表达显著降低（P＜0.05，P＜0.01）；同NMDA损伤组比较，各个干预组细胞生存力提高，培养上清液中LDH释放率降低，凋亡细胞减少，，p-p38MAPK及BAX的表达减少，而BCL-2表达增多（P＜0.05，P＜0.01），以联合干预组效果最为明显。结论 NMDA可诱导皮质神经元损伤，MK801、SB203580对这种损伤具有保护作用，p38MAPK通路可能参与介导MK801的保护作用，其共同机制部分是通过抑制p38MAPK信号通路介导的凋亡相关蛋白实现的。
[Abstract]:Objective to observe the effect of NMDA on primary cultured cortical neuron injury and to observe the effects of NMDA receptor (NMDAR) inhibitor MK801 and p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. To explore the role and mechanism of p38 MAPK signaling pathway in neuronal injury from signal transduction pathway and apoptosis factor. Methods NMDAR specific agonist NMDA was used to induce neuronal injury in vitro. The cortical neurons of neonatal SD rats were cultured for 7 days and were randomly divided into five groups: control group (NMDA50 渭 mol / L) treated with NMDA50 渭 mol / L MK801 (NMDA50 渭 mol / L MK80110 渭 mol / L) and NMDA50 渭 mol / L SB20358010 渭 mol / L (NMDA50 渭 mol / L MK801 and SB203580 10 渭 mol / L each). The neuronal viability was evaluated by immunofluorescence staining against neuron specific dilute alcoholase (NSE), MTT assay and lactate dehydrogenase (LDH) release rate. Acridine orange / ethidium bromide double fluorescent staining was used to observe the morphology and number of apoptosis, and immunocytochemical staining (IHC) and Western blot were used to detect the expression of p-p38MAPKC-2 and Bax in rat cortical neurons. Results compared with the control group, the viability of NMDA injured neurons was significantly decreased, the content of LDH in culture supernatant was increased significantly, and the expression of p-p38 MAPKnBAX protein was significantly increased in apoptotic cells. The expression of BCL-2 protein was significantly decreased (P < 0.05, P < 0.01), the cell viability was increased, the LDH release rate was decreased, and the expression of p-p38 MAPK and Bax was decreased in the culture supernatant. The expression of BCL-2 increased (P < 0.05, P < 0.01), especially in the combined intervention group. Conclusion NMDA can induce cortical neuron injury. MK801 / SB203580 may play a protective role in mediating MK801. The common mechanism is partly through inhibition of apoptosis-related protein mediated by p38 MAPK signaling pathway. [WT5HZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ]
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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