Homer1a在NMDA神经元损伤中的作用及其与nNOS相关性研究

发布时间：2018-06-19 23:53

本文选题：神经元 + 脑创伤　；参考：《第四军医大学》2013年博士论文

【摘要】：创伤性脑损伤(Traumatic brain injury, TBI)是人类致伤致残的主要原因之一，其可分为原发性脑损伤和继发性脑损伤，其中继发性脑损伤是导致患者死亡的重要原因。TBI早期谷氨酸大量释放，作用于各类谷氨酸受体而发挥兴奋性神经毒作用，引起Ca2+通道持续开放致神经元凋亡，是加重继发性损伤的重要原因。谷氨酸与配体门控型Ca2+通道NMDA受体结合后激活该受体，允许Ca2+大量通过，并且由于NMDAR失活缓慢,致使细胞内Ca2+浓度升高上千倍，加重继发性损害，因此研究如何降低NMDA受体引起的神经元损伤，具有显著的临床意义。 Homer1a分子属于Homer蛋白家族，是一个作用广泛的即早基因(ImmediateEarly Gene)，和多种疾病的发生发展都有紧密联系，能够调控多种细胞分子功能。作为突触后致密物质（PSD）的一员，Homer1a可以调控突触后致密物之间的信号转导，是一个重要的钙调节蛋白，它也可以通过物理替代相应结构起到负向调节作用，并且Homer1a能与Shank、NMDA、nNOS等分子发生相互作用。目前Homer1a与NMDAR所致损伤的关系以及和NMDAR下游分子通路的关系尚不清楚，由于NMDA受体在继发性脑损伤中的重要作用，研究Homer1a在NMDA受体所致神经元损伤中的作用具有深远意义。实验一NMDA动物模型的建立目的建立一种可靠而且稳定的小鼠NMDA脑损伤模型，为进一步研究损伤后脑组织病理生理变化及NMDA损伤机制奠定基础。方法将小鼠随机分为假手术组和NMDA皮层注射损伤组，检测NMDA脑损伤后NSS评分和血清NSE，观察甲苯胺蓝染色变化，阐明小鼠皮层注射NMDA致脑损伤模型的效果。结果小鼠皮层注射NMDA损伤后24h，光镜下观察不同组甲苯胺蓝染色变化发现：假损伤组皮层完整，尼氏体清晰可见，皮层无损伤，损伤组损伤灶周围神经元数量明显减少，染色变浅，皮层完整性受到破坏。皮层注射NMDA后观察小鼠行为发现：NMDA损伤组较假损伤组NSS评分高，说明损伤严重。在损伤后12h，24h， NMDA损伤组小鼠血清中NSE高于假损伤组。结论通过检测脑损伤后NSS评分、血清NSE和甲苯胺蓝染色的变化证实，皮层注射NMDA致脑损伤模型具有明显的损伤作用，是一种简单而有效的在体NMDA脑损伤模型。实验二体外培养小鼠脑皮层神经元NMDA损伤模型的建立目的建立一种可靠、稳定而且有效的离体培养小鼠脑皮层神经元NMDA损伤模型。方法对培养成活一周的原代小鼠脑皮层神经元进行神经元纯度鉴定。神经元培养成活后11-15d，按照Koh等的方法，进行离体培养神经元NMDA损伤。将神经元随机分为假损伤对照组和NMDA损伤组，通过Hoechst染色,检测细胞内ROS的水平以及细胞LDH释放，明确神经元NMDA损伤的效果。结果对培养的神经元进行NF200染色显示：培养的神经元纯化率较高。神经元在NMDA损伤后0h，各组间的LDH浓度无明显变化；伤后6h~24h，与就假损伤对照组相比，损伤组的LDH浓度高。进行NMDA损伤后24h，与对照组相比，细胞的凋亡显著增加，ROS荧光亮度显著升高。结论我们通过检测培养液乳酸脱氢酶（Lactate dehydrogenase，LDH）水平、ROS染色、以及Hoechst染色的变化证明，NMDA对神经元有确切的致伤作用。该模型是一种简单而且有效的离体皮层神经元NMDA损伤模型，为研究NMDA所致神经元死亡相关分子机制等提供了一个可靠、有效平台。实验三NMDA损伤（在体与离体）后Homer1a蛋白表达目的通过在体及离体实验，了解NMDA脑损伤后Homer1a表达变化。方法将小鼠或细胞分为假损伤对照组和NMDA损伤组，在NMDA脑损伤模型（在体与离体）基础上，采用免疫组织化学法、免疫印迹法（Western Blotting）和PCR法检测NMDA脑损伤后Homer1a蛋白以及mRNA的变化。结果在体模型中使用NMDA注射皮层后24h，使用免疫组化染色法观察周边脑组织中Homer1a蛋白表达，发现NMDA注射组表达Homer1a蛋白的阳性神经元数高于对照组。在离体NMDA脑损伤模型中，用免疫印迹法检测Homer1a蛋白在NMDA损伤后的表达变化，发现NMDA细胞损伤后6h至24h，Homer1a蛋白表达量明显增加。用普通PCR方法检测Homer1a mRNA，发现相比对照组，NMDA损伤组Homer1a mRNA于损伤后1h、3h表达明显增加。结论Homer1a蛋白和mRNA水平在NMDA损伤后增加，这提示Homer1a可能在NMDA脑损伤中发挥着重要的作用，这种表达水平的增加可能与神经元自身保护机制有关。实验四NMDA损伤对Homer1a K/O小鼠的作用目的通过在体实验，明确NMDA损伤对Homer1a K/O小鼠的作用。方法首先对Homer1a基因敲除鼠进行筛选与鉴定，后将小鼠分为2个组，KO（Knock Out）小鼠组和WT（Wild Type）小鼠组，建立小鼠NMDA脑损伤模型，用甲苯胺蓝染色计算梗死面积、对小鼠进行神经功能学评分（NSS），以及检测血清NSE，明确Homer1aK/O小鼠和WT小鼠的对NMDA损伤作用效应的区别。结果我们成功杂交出了Homer1a基因敲除鼠，普通PCR结果显示，基因敲除小鼠有效，无Homer1a基因表达，并且有Homer1c基因表达。小鼠皮层注射NMDA后24h，尼氏染色计算梗死面积后发现Homer1a K/O组较WT组脑皮层损伤重；NSS评分提示在12h、24h，Homer1aK/O组较WT组评分高；血清NSE含量检测提示损伤后24h，Homer1a K/O组血清中NSE较WT组高。结论这些结果提示，Homer1a基因敲除加重了NMDA引起的脑损伤，Homer1a基因对NMDA脑损伤具有保护作用。实验五体外模型中过表达Homer1a对NMDA神经元损伤的影响目的前面的实验结果提示，敲除小鼠Homer1a基因加重NMDA脑损伤，推论Homer1a基因对NMDA脑损伤有保护作用，但仍需进一步细胞模型中得到证实。方法进行脑皮层神经元原代培养后，转染LV-Homer1a，鉴定表达后，将细胞分为对照损伤组，空载体组，Homer1a过表达组，建立NMDA损伤模型，观察细胞细胞死亡率，进行Hoechst染色分析以及损伤后LDH值测定，Western-Blot进行损伤后p-Caspase-3分析。结果通过使用过表达Homer1a的慢病毒载体（LV-Homer1a）转染神经元发现过表达Homer1a成功，能够明显检测出外源性的Homer1a。用慢病毒载体（LV-Homer1a）转染神经元，造模后24h，对各组神经元进行Hoechst染色，发现转染LV-Homer1a降低了凋亡细胞比例和，降低了p-Caspase3表达。LDH结果提示伤后6h、12h、24h，与对照损伤组、LV-Vector损伤组比，LV-Homer1a的LDH浓度较低。结论这些结果提示，离体试验中，Homer1a能够降低凋亡细胞的比例，保护神经元，也验证了在体试验的结果。实验六Homer1a对NMDAR致损伤功能的影响以及nNOS的活性影响目的前面的实验结果证实，Homer1a对NMDA所致损伤后的神经元具有直接保护作用，但具体的机制尚不清楚。本实验拟在离体模型中研究损伤后Homer1a对NMDAR下游通路的影响，以及Homer1a对nNOS活性的影响。方法脑皮层神经元原代培养成活后转染LV-Homer1a，建立NMDA损伤模型，随机将细胞分为NMDA损伤组、空载体组、转染LV-Homer1a组，观察细胞内产生的ROS，Ca2+、并进行全细胞膜片钳记录，用Western Blot检测各组中细胞内p-nNOS、 p-ERK、 p-CREB的表达。结果神经元NMDA损伤后24h，与对照组相比，转染LV-Homer1a降低了ROS生成和NMDA引起的钙离子内流。转染LV-Homer1a降低了神经元NMDA受体的电流峰值。Western Blot结果提示：转染LV-Homer1a后降低NMDAR过度激活导致的ERK，CREB，nNOS的激活。结论这些结果提示，离体神经元转染LV-Homer1a后，改变了NMDAR的属性，降低了其通透性，减弱其下游通路的过度激活，这为进一步阐明了Homer1a蛋白对NMDA脑损伤的保护机制奠定了基础。实验七Homer1a对神经元中NMDAR以及NMDAR复合体的影响目的前面的实验结果证实，Homer1a对NMDA所致神经元损伤具有直接保护作用，且改变了NMDA属性以及其下游通路的活性，但是具体的机制尚不清楚。我们推测，Homer1a可能是通过调控NMDAR本身分布以及NMDAR和相关的分子集团结合发挥保护作用。方法通过向HEK293T细胞转染NR1NR2B受体以及Homer1a，进行膜片钳记录，按照转染的分子不同分为空载体组、NR1/NR2B组、NR1/NR2B/Con组、NR1/NR2B/Homer1a组，研究Homer1a对NMDA受体功能的直接影响。在神经元转染LV-Homer1a后，将细胞分为空载体组、过表达Homer1a组，通过提取提纯膜蛋白，，检测Homer1a对NMDA受体亚基NR2B分布的影响，通过免疫共沉淀，检测转染LV-Homer1a对NMDA受体亚基NR2B与nNOS蛋白结合的影响。结果在293T细胞上我们成功过表达了NR1NR2B，且具有受体活性，同时转染LV-Homer1a发现，Homer1a对293T细胞上的NR1/NR2B受体的峰值无明显影响。体外培养神经元转染LV-Homer1a72h，发现转染LV-Homer1a后降低膜上NR2B分布。通过对NR2B以及nNOS进行免疫沉淀发现，转染LV-Homer1a降低了NR2B和nNOS之间结合。结论这些结果提示，Homer1a能够促进NMDAR亚基NR2B的内陷以及破坏NR2B,nNOS之间的结合，这两点可能是Homer1a降低NMDA神经元损伤的重要机理。
[Abstract]:Traumatic brain injury (TBI) is one of the main causes of human disability, which can be divided into primary brain injury and secondary brain injury. Secondary brain injury is an important cause of death in patients with.TBI release of glutamic acid at early stage, acting on all kinds of glutamate receptors and exerting excitatory neurotoxicity. The sustained and open neuronal apoptosis of the Ca2+ channel is an important cause of secondary injury. The binding of glutamic acid to the ligand gated Ca2+ channel NMDA receptor activates the receptor, allowing the Ca2+ to pass through, and the increase of the concentration of Ca2+ in the cells increases thousands of times and aggravates secondary damage due to the slow inactivation of NMDAR, thus how to decrease the study. NMDA receptor induced neuronal damage has significant clinical significance.
Homer1a is a family of Homer protein, which is a widely used ImmediateEarly Gene, which is closely related to the occurrence and development of various diseases and can regulate the function of many kinds of cell molecules. As a member of the postsynaptic dense substance (PSD), Homer1a can regulate the signal transduction between the postsynaptic densification and is an important factor. The calcium regulatory protein can also play a negative regulatory role by replacing the corresponding structure by physics, and Homer1a can interact with Shank, NMDA, nNOS and other molecules. The relationship between Homer1a and NMDAR induced damage and the relationship with the downstream pathway of the downstream NMDAR is unclear, due to the important role of NMDA receptor in secondary brain damage. It is of great significance to study the role of Homer1a in NMDA receptor induced neuronal damage. Experiment 1 NMDA animal model was established.
Objective to establish a reliable and stable model of NMDA brain injury in mice, and to lay the foundation for further study of the pathophysiological changes of brain tissue and the mechanism of NMDA injury after injury. Methods mice were randomly divided into sham operation group and NMDA cortex injuried group. The NSS score and serum NSE were detected after NMDA brain injury, and the changes of toluidine blue were observed. The effect of NMDA induced brain injury in the cortex of the mice was given. Results the cortex was injected with 24h after NMDA injury. The changes of toluidine blue in different groups were observed under light microscope: the cortex of the false injury group was complete, the Nissl body was clearly visible, the cortex was not damaged, the number of neurons around the lesion was significantly reduced, the staining was shallow, and the cortex integrity was affected. After the injury, the mouse behavior was observed after the cortex injection of NMDA: the NMDA injury group was higher than the false injury group, which indicated that the injury was serious. The serum NSE of the mice in the 12h, 24h, NMDA injury group was higher than that of the false injury group after the injury. Conclusion the changes of NSS score after the detection of brain injury, the changes of serum NSE and toluidine blue staining proved that the cortex injection of NMDA caused brain damage. It is a simple and effective in vivo NMDA brain injury model.
Experiment two establishment of NMDA damage model of mouse cortical neurons in vitro
Objective to establish a reliable, stable and effective NMDA damage model for the cultured mouse cortical neurons in vitro. Methods the neuron purity of the primary mouse cortical neurons cultured for one week was identified. The neuron culture was 11-15d, and the neuron NMDA damage in vitro was carried out according to the method of Koh. The neurons were randomly divided. For the false injury control group and the NMDA injury group, the level of ROS in the cells and the release of LDH were detected by Hoechst staining. The effect of NMDA damage on the neurons was determined. Results the cultured neurons were stained with NF200 to show that the cultured neurons had higher purification rate. There was no obvious change in LDH concentration in each group after the injury of NMDA. After 6h~24h, the concentration of LDH in the injured group was higher than that of the control group. After NMDA injury, 24h, compared with the control group, the apoptosis of the cells increased significantly and the ROS fluorescence luminance increased significantly. Conclusion we have proved that the NMDA pairs were detected by detecting the level of Lactate dehydrogenase (LDH), ROS staining, and Hoechst staining. This model is a simple and effective model of NMDA damage in isolated cortical neurons, which provides a reliable and effective platform for the study of the molecular mechanism of neuronal death induced by NMDA.
Experiment three the expression of Homer1a protein after NMDA injury (in vitro and in vivo).
Objective to understand the changes of Homer1a expression after NMDA brain injury in vivo and in vitro. Methods mice or cells were divided into false damage control group and NMDA injury group. On the basis of NMDA brain damage model (in vivo and in vitro), immunohistochemistry, Western Blotting and PCR method were used to detect the Homer1a protein after NMDA brain injury. Results in the body model, 24h was injected after the injection of NMDA in the body model, and the expression of Homer1a protein in the peripheral brain tissue was observed by immunohistochemical staining. The number of positive neurons expressing Homer1a protein in NMDA injection group was higher than that of the control group. In the isolated NMDA brain injury model, the immunoblotting method was used to detect the Homer1a protein after NMDA injury. The expression of 6h to 24h and the expression of Homer1a protein increased significantly after NMDA cell injury. Homer1a mRNA was detected by common PCR method. It was found that the Homer1a mRNA of NMDA damage group was 1h after injury, and the 3H expression increased obviously. This increase in expression level may be related to the mechanism of neuronal self protection.
Effect of Experiment four NMDA damage on Homer1a K/O mice
Objective to determine the effect of NMDA injury on Homer1a K/O mice in vivo. Methods first of all, the Homer1a gene knockout mice were screened and identified. Then the mice were divided into 2 groups, KO (Knock Out) mice and WT (Wild Type) mice. The mouse NMDA brain damage model was established. The infarct area was calculated with toluidine blue staining, and the mice were nervous. The functional score (NSS), and the detection of serum NSE, make clear the difference between the Homer1aK/O mice and the WT mice. Results we successfully hybridized the Homer1a gene knockout mice. The common PCR results showed that the gene knockout mice were effective, no Homer1a gene expression, and the Homer1c gene expression. The mouse cortex was injected with NMDA 24h, Nei. It was found that Homer1a K/O group was more severe than group WT, and NSS score was higher in 12h, 24h, Homer1aK/O group than that in group WT, and the serum NSE content detection suggested that the serum levels of 24h and Homer1a K/O group were higher than those in the group of WT. Conclusion these results suggest that the gene knockout aggravates the brain damage caused by the gene knockout. The gene has protective effects on NMDA brain damage.
Effect of overexpression of Homer1a in vitro on NMDA neuron injury in experiment five
The previous experimental results suggest that the Homer1a gene in the knockout mice aggravates NMDA brain damage and that the Homer1a gene has a protective effect on the brain damage of NMDA, but it still needs to be confirmed in the further cell model. After the primary culture of the cortical neurons, the transfected LV-Homer1a was divided into the control injury group and the empty body group after the transfection of LV-Homer1a. Homer1a overexpression group, establish NMDA damage model, observe cell death rate, analyze Hoechst staining and determine LDH value after injury, p-Caspase-3 analysis after Western-Blot damage. The result can be detected by transfection of Homer1a based lentivirus vector (LV-Homer1a) to the expression of Homer1a, which can be detected obviously. Exogenous Homer1a. transfected neurons with lentivirus vector (LV-Homer1a), and then 24h, Hoechst staining was performed on each group of neurons. It was found that transfection of LV-Homer1a reduced the proportion of apoptotic cells and decreased the p-Caspase3 expression.LDH results, suggesting 6h, 12h, 24h, compared with the control injury group, LV-Vector injury group, and low concentration of LV-Homer1a. Conclusion these results suggest that Homer1a can reduce the proportion of apoptotic cells and protect neurons in vitro, and also verify the results of in vivo experiments.
Experiment six the effect of Homer1a on NMDAR induced damage and the activity of nNOS.
The previous experimental results confirm that Homer1a has a direct protective effect on the neurons injured by NMDA, but the specific mechanism is still unclear. This experiment is intended to study the effect of Homer1a on the downstream pathway of NMDAR after injury and the effect of Homer1a on the activity of nNOS. LV-Homer1a, the NMDA damage model was established, and the cells were randomly divided into NMDA injury group, empty body group, transfected LV-Homer1a group, and observed the ROS, Ca2+, and whole cell patch clamp recording. The expression of p-nNOS, p-ERK, p-CREB in each group was detected by Western Blot. Results the neuron NMDA was injured and transfected compared with the control group. LV-Homer1a reduced the influx of calcium ions caused by ROS formation and NMDA. Transfection of LV-Homer1a reduced the peak current peak of NMDA receptor.Western Blot results, suggesting that the transfection of LV-Homer1a to reduce ERK, CREB, nNOS activation caused by NMDAR overactivation. The property, which reduces its permeability and weakens the over activation of its downstream pathway, lays the foundation for further clarifying the protective mechanism of Homer1a protein to NMDA brain damage.
Effect of experiment seven Homer1a on NMDAR and NMDAR complex in neurons
The previous experimental results confirm that Homer1a has a direct protective effect on NMDA induced neuronal damage and changes the NMDA property and its downstream pathway activity, but the specific mechanism is still unclear. We speculate that Homer1a may play a protective role by regulating the distribution of NMDAR itself and the combination of NMDAR and related molecular groups. Methods through the transfection of NR1NR2B receptor and Homer1a to HEK293T cells, the patch clamp recording was carried out. According to the different transfection molecules, the cells were divided into empty carrier group, NR1/NR2B group, NR1/NR2B/Con group and NR1/NR2B/Homer1a group, to study the direct effect of Homer1a on the function of NMDA receptor. After transfection of LV-Homer1a, the cells were divided into empty carrier group and overexpressed Homer. In group 1a, the effect of Homer1a on the distribution of NMDA receptor subunit NR2B was detected by extracting purified membrane protein, and the effect of LV-Homer1a on the binding of NMDA receptor subunit NR2B to nNOS protein was detected by immunoprecipitation. The results showed that NR1NR2B was successfully expressed on 293T cells, with receptor activity, and LV-Homer1a discovery was transfected, Homer1a against 29 The peak value of NR1/NR2B receptor on 3T cells was not significantly affected. The transfection of LV-Homer1a72h to neurons in vitro was found to reduce the distribution of NR2B on the membrane. By immunoprecipitation of NR2B and nNOS, the transfection of LV-Homer1a reduced the binding between NR2B and nNOS.
Conclusion these results suggest that Homer1a can promote the collapse of NMDAR subunit NR2B and destroy the binding of NR2B and nNOS, which may be an important mechanism for Homer1a to reduce the damage of NMDA neurons.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R651.15

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