间歇低氧后常氧培养不同时间胎鼠海马神经元细胞HMGB1和NMDA表达变化
发布时间：2018-09-17 10:09
【摘要】：背景与目的:阻塞性睡眠呼吸暂停(OSA)引起并发症可涉及多个系统,对于中枢神经系统(CNS)来说成人和儿童均引起认知功能障碍,包括嗜睡、注意力下降、记忆力减退、情绪和表达障碍。间歇低氧(IH)是OSA的重要病理生理特征之一,主要通过海马CA1区域神经元的凋亡引起认知功能障碍。CNS IH可引发活性氧簇过度产生,导致炎性物质大量产生致神经元凋亡和坏死并加重OSA相关认知功能障碍。因此本研究通过原代培养海马神经元5-7 d,并进行分组,经低氧暴露后测定不同分组炎症因子的表达水平,从而探讨神经炎症在间歇低氧引起的神经认知障碍中的作用。实验方法:采用孕16-18d SD孕鼠的胎鼠分离其海马神经元细胞,原代培养5-7天后,进行实验。首先进行细胞形态学观察,分别于不同时间采用倒置相差显微镜观察细胞形态,采集细胞形态图像;采用结构性微管相关蛋白-2(MAP-2)标记神经元胞体和树突,DAPI染核进行免疫荧光染色鉴定,在荧光显微镜下观察细胞、采集图像。挑选细胞长势良好、无污染的细胞,随机将细胞分为常氧对照组和间歇低氧组。采用天津医科大学总医院呼吸科仿真系统1.0设置低氧/再氧合循环模式,低氧混合气含1.5%O2、93.5%N2、5%CO2,正常氧混合气含21%O2、74%N2、5%CO2,暴露频率6次/h,暴露时间为4h。再根据暴露后继续常氧培养不同时间将间歇低氧组细胞分为2h组,4h组,6h组,8h组。间歇低氧暴露结束后提取对照组和间歇低氧组海马神经元细胞总mRNA,采用Real Time PCR测定两组细胞高迁移率族蛋白1(HMGB1)mRNA相对表达水平。待常氧培养结束后提取细胞总mRNA和细胞总蛋白水平,利用Real Time PCR和Western blot方法检测对照组和各培养组HMGB1和N-甲基-D-天冬氨酸(NMDA)相对表达水平。采用SPSS和GraphPad Prism进行统计分析。结果:(1)培养的海马神经元细胞4h后贴壁明显,少数细胞开始长出1-2个突起,细胞折光性比较强,未见明显细胞核。神经元细胞接种24h后大多数细胞已贴壁,细胞呈圆形或椭圆形且周围有光晕,单个均匀分布,可见有3个突起或多突起的神经元。培养3d后神经元突起增多并变长,胞体变大,细胞聚集成团,可见突起连接成稀疏的网状结构。5d后神经元胞体成三角形、圆形或多边形,突起形成较密集的网络。7d后神经元生长良好,胞体清晰,突起增粗形成致密的神经网络。对培养5-7d的海马神经元细胞采用神经元特异性标志物Map-2和细胞核DAP I染色后在倒置荧光显微镜采用不同激发波长观察,可见绿色神经元突起和胞体以及蓝色荧光的细胞核,两图重叠后可计算神经元所占比例,计算阳性细胞比例在90%以上。(2)IH组和对照组相比较,IH组HMGB1 mRNA相对表达量是对照组的3.203倍,IH组HMGB1 mRNA表达水平明显增高(P0.05)。IH组暴露后继续培养不同时间各组之间HMGB1mRNA相对表达水平差异明显(P0.05),其中4H mRNA相对表达量水平最高。约为对照组的8.142倍(P0.0001),2h HMGB1表达约为对照组的2.836倍(P0.05),6h约为对照组的0.669倍,与对照组相比表达量减少(P0.01),8h约为对照组的0.338倍,与对照组相比表达量减少(P0.01)。与2H相对表达水平相比,4H表达量明显增多(P0.05);6H与2H相比,6H相对表达水平增加(P0.05);8H与2H相比,8H相对表达水平增高(P0.05);6H与4H相比,6H相对表达水平明显增高(P0.05);8H与4H相比,8H相对表达水平增高(P0.05)。(3)IH组暴露后继续培养不同时间各组之间NMDA mRNA相对表达水平差异明显(P0.05),随着正常氧培养时间的延长,各组NMDA mRNA相对表达水平逐渐增高(P0.05)。2h与对照组相比相对表达水平明显升高(P0.01),4h与对照组相比相对表达量增加(P0.001),6h与对照组相比表达水平明显增加,8h与对照组相比表达量增加(P0.001)。(4)IH组暴露后继续培养不同时间海马神经元细胞内HMGB1蛋白表达水平差异明显(P0.05),与对照组相比4h蛋白表达水平最高,2h蛋白表达与对照组相比增加,6h蛋白表达水平较对照组相比明显减少,8h蛋白表达水平与对照组相比明显降低。IH组暴露后继续培养不同时间海马神经元细胞内NMDA蛋白表达水平存在明显差异(P0.05),随着暴露后常氧培养时间延长,海马神经元细胞内NMDA蛋白表达明显增多。8h海马神经元细胞表达NMDA蛋白量最多。结论:神经炎症在OSA IH引起的神经认知功能障碍中发挥重要作用,IH可引起神经炎症因子HMGB1表达。说明神经炎症和损伤的神经元之间形成恶性循环,对神经元造成“二次打击”。即使IH暴露撤除神经元的损伤也持续存在。濒临死亡的神经元持续释放HMGB1对于形成和维持这个恶性循环有重要意义。即使始动因素IH停止后,炎性损伤还能继续。IH可以引起神经炎症,但IH不是维持神经炎症的必须条件。随着炎症持续时间延长,因子HMGB1分泌减少,但NMDA明显增多,表明可能HMGB1和NMDA可能发挥协同作用,引起神经元凋亡和功能障碍。
[Abstract]:BACKGROUND AND OBJECTIVE: Obstructive sleep apnea (OSA) can cause complications involving multiple systems. For the central nervous system (CNS), cognitive impairment occurs in adults and children, including lethargy, decreased attention, memory loss, mood and expression disorders. Intermittent hypoxia (IH) is one of the important pathophysiological characteristics of OSA, mainly through. Neuronal apoptosis in hippocampal CA1 region leads to cognitive impairment. CNS IH can induce excessive production of reactive oxygen species (ROS) clusters, resulting in excessive production of inflammatory substances that induce neuronal apoptosis and necrosis and aggravate OSA-related cognitive impairment. Methods: The hippocampal neurons of SD pregnant rats from 16 to 18 days of gestation were isolated and cultured for 5 to 7 days. Cell morphology was observed under microscope and cell morphology images were collected. Cells were labeled with structural microtubule-associated protein-2 (MAP-2) and identified by immunofluorescence staining with DAPI staining. Cells were observed under fluorescence microscope and collected images. Hypoxia group. Hypoxia/reoxygenation cycle mode was set up in the Simulation System 1.0 of Respiratory Department of Tianjin Medical University General Hospital. Hypoxia mixture contained 1.5% O2, 93.5% N2, 5% CO2, normal oxygen mixture contained 21% O2, 74% N2, 5% CO2. The exposure frequency was 6 times/h and the exposure time was 4 hours. Total mRNA of hippocampal neurons in control group and intermittent hypoxia group was extracted after intermittent hypoxic exposure, and the relative expression levels of high mobility group protein 1 (HMGB1) mRNA in the two groups were determined by Real Time PCR. The relative expression levels of HMGB1 and N-methyl-D-aspartate (NMDA) in the control group and the cultured groups were detected by lot method. SPSS and GraphPad Prism were used for statistical analysis. Most of the cells adhered to the wall 24 hours after inoculation. The cells were round or elliptical with halo around them. There were three or more neurites in the cells. After 3 days of culture, the neurites increased and lengthened, the cell bodies became larger, the cells aggregated into clusters, and the neurite bodies formed triangular and round structures. After 7 days, the neurons grew well, the soma was clear, and the processes were thickened to form a dense neural network. The hippocampal neurons cultured for 5-7 days were stained with Map-2, a neuron-specific marker, and DAP I of the nucleus. Different excitation wavelengths were observed under the inverted fluorescence microscope. Compared with the control group, the relative expression of HMGB1 mRNA in IH group was 3.203 times higher than that in control group, and the expression level of HMGB1 mRNA in IH group was significantly higher than that in control group (P 0.05). The relative expression level of HMGB1 mRNA in each group was significantly different (P 0.05), among which the relative expression level of 4H mRNA was the highest, about 8.142 times (P 0.0001), 2 hours HMGB1 expression was 2.836 times (P 0.05), 6 hours HMGB1 expression was 0.669 times (P 0.01), 8 hours was 0.338 times (P 0.01), and compared with the control group. Compared with 2H, 4H expression increased significantly (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), and 8H relative expression increased (P 0.05) compared with 4H relative expression level. Relative expression level of NMDA mRNA was significantly different between groups at different time after exposure (P 0.05). With the prolongation of normal oxygen culture time, the relative expression level of NMDA mRNA gradually increased (P 0.05). The relative expression level of NMDA mRNA in each group was significantly higher than that in the control group at 2 h (P 0.01), 4 h (P 0.001), 6 h and 6 h, respectively. The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly higher than that of control group at 8 hours (P 0.001). (4) The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). Compared with control group, the expression level of HMGB1 protein was the highest at 4 hours, the expression level of 2H protein was higher than that of control group, and the expression level The expression level of NMDA protein in hippocampal neurons of IH group was significantly lower than that of control group at 8 hours after exposure. The expression level of NMDA protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). The expression of NMDA protein in hippocampal neurons increased significantly with the prolongation of normoxic culture time after exposure. Conclusion: Neuritis plays an important role in neurocognitive impairment induced by OSA-IH, and IH can induce the expression of neuroinflammatory factor HMGB1. This indicates that there is a vicious circle between neurons with neuroinflammation and injury, which can cause a "second blow" to neurons. Continuous release of HMGB1 from dying neurons is important for the formation and maintenance of this malignant cycle. Inflammatory damage can continue even after the initiating factor IH ceases. IH can cause neuroinflammation, but IH is not a necessary condition for maintaining neuroinflammation. As the duration of inflammation prolongs, the secretion of HMGB1 decreases, but NMDA is clear. Significant increase indicates that HMGB1 and NMDA may play a synergistic role in inducing neuronal apoptosis and dysfunction.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766
本文编号：2245536
[Abstract]:BACKGROUND AND OBJECTIVE: Obstructive sleep apnea (OSA) can cause complications involving multiple systems. For the central nervous system (CNS), cognitive impairment occurs in adults and children, including lethargy, decreased attention, memory loss, mood and expression disorders. Intermittent hypoxia (IH) is one of the important pathophysiological characteristics of OSA, mainly through. Neuronal apoptosis in hippocampal CA1 region leads to cognitive impairment. CNS IH can induce excessive production of reactive oxygen species (ROS) clusters, resulting in excessive production of inflammatory substances that induce neuronal apoptosis and necrosis and aggravate OSA-related cognitive impairment. Methods: The hippocampal neurons of SD pregnant rats from 16 to 18 days of gestation were isolated and cultured for 5 to 7 days. Cell morphology was observed under microscope and cell morphology images were collected. Cells were labeled with structural microtubule-associated protein-2 (MAP-2) and identified by immunofluorescence staining with DAPI staining. Cells were observed under fluorescence microscope and collected images. Hypoxia group. Hypoxia/reoxygenation cycle mode was set up in the Simulation System 1.0 of Respiratory Department of Tianjin Medical University General Hospital. Hypoxia mixture contained 1.5% O2, 93.5% N2, 5% CO2, normal oxygen mixture contained 21% O2, 74% N2, 5% CO2. The exposure frequency was 6 times/h and the exposure time was 4 hours. Total mRNA of hippocampal neurons in control group and intermittent hypoxia group was extracted after intermittent hypoxic exposure, and the relative expression levels of high mobility group protein 1 (HMGB1) mRNA in the two groups were determined by Real Time PCR. The relative expression levels of HMGB1 and N-methyl-D-aspartate (NMDA) in the control group and the cultured groups were detected by lot method. SPSS and GraphPad Prism were used for statistical analysis. Most of the cells adhered to the wall 24 hours after inoculation. The cells were round or elliptical with halo around them. There were three or more neurites in the cells. After 3 days of culture, the neurites increased and lengthened, the cell bodies became larger, the cells aggregated into clusters, and the neurite bodies formed triangular and round structures. After 7 days, the neurons grew well, the soma was clear, and the processes were thickened to form a dense neural network. The hippocampal neurons cultured for 5-7 days were stained with Map-2, a neuron-specific marker, and DAP I of the nucleus. Different excitation wavelengths were observed under the inverted fluorescence microscope. Compared with the control group, the relative expression of HMGB1 mRNA in IH group was 3.203 times higher than that in control group, and the expression level of HMGB1 mRNA in IH group was significantly higher than that in control group (P 0.05). The relative expression level of HMGB1 mRNA in each group was significantly different (P 0.05), among which the relative expression level of 4H mRNA was the highest, about 8.142 times (P 0.0001), 2 hours HMGB1 expression was 2.836 times (P 0.05), 6 hours HMGB1 expression was 0.669 times (P 0.01), 8 hours was 0.338 times (P 0.01), and compared with the control group. Compared with 2H, 4H expression increased significantly (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), and 8H relative expression increased (P 0.05) compared with 4H relative expression level. Relative expression level of NMDA mRNA was significantly different between groups at different time after exposure (P 0.05). With the prolongation of normal oxygen culture time, the relative expression level of NMDA mRNA gradually increased (P 0.05). The relative expression level of NMDA mRNA in each group was significantly higher than that in the control group at 2 h (P 0.01), 4 h (P 0.001), 6 h and 6 h, respectively. The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly higher than that of control group at 8 hours (P 0.001). (4) The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). Compared with control group, the expression level of HMGB1 protein was the highest at 4 hours, the expression level of 2H protein was higher than that of control group, and the expression level The expression level of NMDA protein in hippocampal neurons of IH group was significantly lower than that of control group at 8 hours after exposure. The expression level of NMDA protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). The expression of NMDA protein in hippocampal neurons increased significantly with the prolongation of normoxic culture time after exposure. Conclusion: Neuritis plays an important role in neurocognitive impairment induced by OSA-IH, and IH can induce the expression of neuroinflammatory factor HMGB1. This indicates that there is a vicious circle between neurons with neuroinflammation and injury, which can cause a "second blow" to neurons. Continuous release of HMGB1 from dying neurons is important for the formation and maintenance of this malignant cycle. Inflammatory damage can continue even after the initiating factor IH ceases. IH can cause neuroinflammation, but IH is not a necessary condition for maintaining neuroinflammation. As the duration of inflammation prolongs, the secretion of HMGB1 decreases, but NMDA is clear. Significant increase indicates that HMGB1 and NMDA may play a synergistic role in inducing neuronal apoptosis and dysfunction.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766
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