电磁辐射对原代培养皮层神经元tau蛋白磷酸化的影响及其机制初步探讨

发布时间：2018-07-13 21:02

【摘要】：目的：随着科学技术和信息技术的进步和发展，在人类生存空间中的电磁辐射日益增强，电磁辐射辐照的生物效应、机理和防护已成为人们关注的热点问题。大量证据表明，，电磁辐射辐照以中枢神经系统(central nervous system，CNS)的损伤最为显著，电磁辐射辐照致中枢神经系统损伤的早期表现是神经系统功能障碍，主要表现是学习记忆功能障碍，而电磁辐射辐照导致神经系统功能障碍的重要原因之一是神经元的损伤。tau蛋白主要存在于神经元中的一组微管相关蛋白，其生物学功能是调节微管动力、突出运输和神经突生长。病理状态下，tau蛋白的异常过度磷酸化导致微管亲和性降低，进而导致微管不稳定和轴浆运输下降，从而引发神经退行性病理改变，最终引起学习记忆功能障碍。细胞周期素依赖性蛋白激酶5(cyclin dependent kinase5,CDK5)和糖原合成激酶-3β(glycogen synthase kinase-3β，GSK-3β)是最重要的调节tau蛋白磷酸化的蛋白激酶。本研究旨在研究电磁辐射对神经元中tau蛋白磷酸化的影响及初步探讨其相关机制，为电磁辐射辐照致中枢神经系统损伤机制提供新的研究线索。方法：(1)采用平均功率密度为90mW/cm2电磁波辐照原代培养的SD大鼠皮层神经元10min建立神经元损伤模型。(2)采用CCK-8检测试剂盒，检测电磁辐射辐照后神经元活性变化。(3)采用免疫细胞化学(ICC)方法，分析电磁辐射辐照后神经元突出长度变化情况。(4)采用Western-blot技术，检测tau蛋白磷酸化位点ser199/202、ser396、ser404磷酸化水平。(5)采用Western-blot技术，检测CDK5抑制剂Roscovitine、GSK-3β抑制剂Licl、calpain抑制剂PD150606单独或联合预处理对tau蛋白磷酸化的影响。(6)采用免疫细胞化学(ICC)方法，分析CDK5抑制剂Roscovitine、GSK-3β抑制剂Licl、calpain抑制剂PD150606单独或联合预处理后对神经元突出损伤的影响。结果：(1)电磁辐射辐照后可导致神经元活性在辐照后6h、12h显著降低。(2)电磁辐射辐照后1h、3h，神经元突出长度明显缩短。(3)原代培养大鼠皮层神经元tau蛋白ser404位点磷酸化程度在电磁辐射辐照后1h、3h明显增强，而ser199/202、ser396位点磷酸化程度变化不明显。(4)CDK5抑制剂Roscovitine、GSK-3β抑制剂Licl、calpain抑制剂PD150606单独或联合预处理对tau蛋白ser404磷酸化位点的过度磷酸化有明显抑制作用。(5)CDK5抑制剂Roscovitine、GSK-3β抑制剂Licl、calpain抑制剂PD150606单独或联合预处理可明显拮抗电磁辐射辐照引起的神经元突出长度的缩短。结论：电磁辐射辐照可引起神经元明显损伤并可引起神经元tau蛋白ser404位点过度磷酸化，同时CDK5、GSK-3β和calpain参与其中。这些结果为研究电磁辐射辐照致神经元损伤机理以及防护措施提供了新的研究线索。
[Abstract]:Objective: with the progress and development of science and technology and information technology, the electromagnetic radiation in human living space is increasing day by day. The biological effect, mechanism and protection of electromagnetic radiation have become a hot issue that people pay attention to. A large number of evidences show that the damage of central nervous system induced by electromagnetic radiation is the most obvious, and the early manifestation of electromagnetic radiation is the dysfunction of nervous system, which is mainly characterized by learning and memory dysfunction. One of the important causes of nervous system dysfunction caused by electromagnetic radiation is the injury of neurons. Tau protein mainly exists in a group of microtubule-associated proteins in neurons, whose biological function is to regulate microtubule dynamics. Protruding transport and neurite growth. Abnormal hyperphosphorylation of tau leads to the decrease of microtubule affinity, which leads to the instability of microtubules and the decrease of axonal transport, which leads to neurodegenerative pathological changes and ultimately to learning and memory dysfunction. Cyclin dependent protein kinase 5 (cyclin dependent kinase5 (CDK5) and glycogen synthesis kinase 3 尾 (glycogen synthase kinase-3 尾 (GSK-3 尾) are the most important protein kinases that regulate the phosphorylation of tau protein. The purpose of this study was to investigate the effect of electromagnetic radiation on the phosphorylation of tau protein in neurons and its related mechanism, and to provide a new clue for the mechanism of central nervous system injury induced by electromagnetic radiation. Methods: (1) 10min of primary cultured SD rat cortical neurons was irradiated with an average power density of 90 MW / cm ~ 2. (2) CCK-8 detection kit was used. (3) immunocytochemistry (ICC) method was used to analyze the changes of neuronal outburst length after electromagnetic radiation. (4) Western-blot technique was used. The phosphorylation level of tau protein was detected at ser199- / 202ser396nser404. (5) the effect of pretreatment of CDK5 inhibitor Roscovitine GSK-3 尾 inhibitor, Liclcalpain inhibitor PD150606, on the phosphorylation of tau protein was detected by Western-blot. (6) Immunocytochemistry was used to study the effect of Phosphorylation of CDK5 inhibitor Roscovitine Gluta-3 尾 inhibitor PD150606 on the phosphorylation of tau protein. The effects of CDK5 inhibitor Roscovitine glutathione (GSK-3 尾) inhibitor Liclcalpain inhibitor PD150606 alone or in combination on neuronal protrusion injury were analyzed. Results: (1) the neuronal activity decreased significantly 6 h after irradiation and 12 h after irradiation. (2) the neuronal protruding length was significantly shortened at 1 h and 3 h after irradiation. (3) the phosphorylation of the ser404 site of tau protein in primary cultured rat cortical neurons was significantly shortened. The degree of radiation increased significantly at 1 h after irradiation, and increased at 3 h after electromagnetic radiation. However, the phosphorylation level at the site of ser1999 / 202 was not obvious. (4) the CDK5 inhibitor Roscovitine GSK-3 尾 inhibitor Liclcalpain inhibitor PD150606 alone or in combination had a significant inhibitory effect on the hyperphosphorylation of ser404 phosphorylation site of tau protein. (5) the CDK5 inhibitor Roscovitine GSK-3 尾 inhibitor Liclcalpain inhibitor has a significant inhibitory effect on the hyperphosphorylation of ser404 phosphorylation site. (5) the CDK5 inhibitor Roscovitine GSK-3 尾 inhibitor Liclcalpain inhibitor has a significant inhibitory effect on ser404 phosphorylation. PD150606 pretreatment alone or in combination could significantly antagonize the shortening of neuronal protruding length induced by electromagnetic radiation. Conclusion: electromagnetic radiation can cause obvious damage to neurons and excessive phosphorylation of ser404 site of tau protein in neurons, in which CDK5 GSK-3 尾 and calpain are involved. These results provide a new clue for studying the mechanism of neuron damage induced by electromagnetic radiation and protective measures.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R142

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