電磁輻射對(duì)原代培養(yǎng)皮層神經(jīng)元tau蛋白磷酸化的影響及其機(jī)制初步探討
發(fā)布時(shí)間：2018-07-13 21:02
【摘要】：目的：隨著科學(xué)技術(shù)和信息技術(shù)的進(jìn)步和發(fā)展，在人類生存空間中的電磁輻射日益增強(qiáng)，電磁輻射輻照的生物效應(yīng)、機(jī)理和防護(hù)已成為人們關(guān)注的熱點(diǎn)問題。大量證據(jù)表明，，電磁輻射輻照以中樞神經(jīng)系統(tǒng)(central nervous system，CNS)的損傷最為顯著，電磁輻射輻照致中樞神經(jīng)系統(tǒng)損傷的早期表現(xiàn)是神經(jīng)系統(tǒng)功能障礙，主要表現(xiàn)是學(xué)習(xí)記憶功能障礙，而電磁輻射輻照導(dǎo)致神經(jīng)系統(tǒng)功能障礙的重要原因之一是神經(jīng)元的損傷。tau蛋白主要存在于神經(jīng)元中的一組微管相關(guān)蛋白，其生物學(xué)功能是調(diào)節(jié)微管動(dòng)力、突出運(yùn)輸和神經(jīng)突生長(zhǎng)。病理狀態(tài)下，tau蛋白的異常過度磷酸化導(dǎo)致微管親和性降低，進(jìn)而導(dǎo)致微管不穩(wěn)定和軸漿運(yùn)輸下降，從而引發(fā)神經(jīng)退行性病理改變，最終引起學(xué)習(xí)記憶功能障礙。細(xì)胞周期素依賴性蛋白激酶5(cyclin dependent kinase5,CDK5)和糖原合成激酶-3β(glycogen synthase kinase-3β，GSK-3β)是最重要的調(diào)節(jié)tau蛋白磷酸化的蛋白激酶。本研究旨在研究電磁輻射對(duì)神經(jīng)元中tau蛋白磷酸化的影響及初步探討其相關(guān)機(jī)制，為電磁輻射輻照致中樞神經(jīng)系統(tǒng)損傷機(jī)制提供新的研究線索。 方法：(1)采用平均功率密度為90mW/cm2電磁波輻照原代培養(yǎng)的SD大鼠皮層神經(jīng)元10min建立神經(jīng)元損傷模型。(2)采用CCK-8檢測(cè)試劑盒，檢測(cè)電磁輻射輻照后神經(jīng)元活性變化。(3)采用免疫細(xì)胞化學(xué)(ICC)方法，分析電磁輻射輻照后神經(jīng)元突出長(zhǎng)度變化情況。(4)采用Western-blot技術(shù)，檢測(cè)tau蛋白磷酸化位點(diǎn)ser199/202、ser396、ser404磷酸化水平。(5)采用Western-blot技術(shù)，檢測(cè)CDK5抑制劑Roscovitine、GSK-3β抑制劑Licl、calpain抑制劑PD150606單獨(dú)或聯(lián)合預(yù)處理對(duì)tau蛋白磷酸化的影響。(6)采用免疫細(xì)胞化學(xué)(ICC)方法，分析CDK5抑制劑Roscovitine、GSK-3β抑制劑Licl、calpain抑制劑PD150606單獨(dú)或聯(lián)合預(yù)處理后對(duì)神經(jīng)元突出損傷的影響。 結(jié)果：(1)電磁輻射輻照后可導(dǎo)致神經(jīng)元活性在輻照后6h、12h顯著降低。(2)電磁輻射輻照后1h、3h，神經(jīng)元突出長(zhǎng)度明顯縮短。(3)原代培養(yǎng)大鼠皮層神經(jīng)元tau蛋白ser404位點(diǎn)磷酸化程度在電磁輻射輻照后1h、3h明顯增強(qiáng)，而ser199/202、ser396位點(diǎn)磷酸化程度變化不明顯。(4)CDK5抑制劑Roscovitine、GSK-3β抑制劑Licl、calpain抑制劑PD150606單獨(dú)或聯(lián)合預(yù)處理對(duì)tau蛋白ser404磷酸化位點(diǎn)的過度磷酸化有明顯抑制作用。(5)CDK5抑制劑Roscovitine、GSK-3β抑制劑Licl、calpain抑制劑PD150606單獨(dú)或聯(lián)合預(yù)處理可明顯拮抗電磁輻射輻照引起的神經(jīng)元突出長(zhǎng)度的縮短。 結(jié)論：電磁輻射輻照可引起神經(jīng)元明顯損傷并可引起神經(jīng)元tau蛋白ser404位點(diǎn)過度磷酸化，同時(shí)CDK5、GSK-3β和calpain參與其中。這些結(jié)果為研究電磁輻射輻照致神經(jīng)元損傷機(jī)理以及防護(hù)措施提供了新的研究線索。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R142
本文編號(hào)：2120731
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R142
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