免疫相关受体PirB在慢性炎症脑神经元突触丢失中的作用
发布时间：2018-07-21 20:48
【摘要】：目的1.探讨内毒素脂多糖诱导的慢性炎症脑神经元和星形胶质细胞PirB的表达变化,突触囊泡素的表达及动物行为学的改变;2.探讨PirB在慢性炎症脑神经元突触丢失和学习记忆功能缺失中的免疫调节作用。 方法正常成年SD大鼠,随机分为实验组和对照组,依赖立体定位技术,在大鼠右侧海马内分别注射LPS和PBS,动物存活30天。用免疫组织化学法检测大鼠脑皮质、海马PirB、突触囊泡素的表达及星形胶质细胞GFAP的表达变化,用图像分析系统检测阳性细胞数目和染色强度；并采用Western blot技术对其进行定量分析。高尔基染色观察大鼠脑神经元结构的改变。免疫荧光双重标记技术,观察PirB阳性细胞与神经元、星型胶质细胞的共存及PirB阳性细胞与突触的关系;Morris水迷宫测试用于评估实验组大鼠的学习和空间记忆能力。 结果GFAP阳性星形胶质细胞分布在整个海马结构、梨状皮质和内嗅皮质、皮质以下的白质如胼胝体等部位。海马内注射PBS的对照组动物,海马、皮质内GFAP阳性的星形胶质细胞展示出静息的形态特征：胞体小,分支细长,染色较淡。海马内注射LPS的实验组动物海马、梨状皮质和内嗅皮质内GFAP阳性星形胶质细胞明显被激活,表现为细胞胞体明显增大,突起变肥大；定量分析表明,LPS处理30天后,海马CA3区和齿状回内GFAP阳性星形胶质细胞数目和光密度OD值明显增加,差异有显著性(P0.01)。 PBS注射组动物脑皮质V层内可见PirB阳性神经元样细胞,呈圆形、卵圆形和锥体形,免疫反应产物呈棕色,主要位于细胞膜上；海马内注射LPS30天后,可见较大的PirB阳性神经元样细胞和较小的PirB阳性神经胶质样细胞主要分布于皮质Ⅳ、V层、海马CA1-CA3区锥体细胞层、齿状回颗粒细胞层,免疫反应产物位于胞质和突起内。定量分析表明,与对照组比较,LPS处理30d后,皮质、海马内PirB阳性神经元的数目和OD值明显增加,差异有显著性(P0.005)。Western blot定量分析：LPS处理30天后,注射侧皮质、海马内PirB蛋白质的表达均明显增加,差异有显著性(P0.001)。PirB分别与MAP-2、GFAP、CDllb的免疫荧光双重标记染色显示：皮质、海马内PirB和MAP-2阳性神经元的胞体存在共定位；PirB与GFAP阳性星形胶质细胞存在部分共定位,实验组海马内GFAP阳性星形胶质细胞明显被激活,活化的星形胶质细胞PirB表达上调,但未见PirB与CDllb阳性染色双标细胞。 SYN阳性产物呈点状棕色颗粒,在PBS处理的大鼠海马主要分布于海马CA1区的始层、辐射层及分子层,CA3区阿蒙氏角神经纤维终末以及齿状回的非细胞层。与对照组相比,LPS处理的大鼠脑海马CA1区、齿状回内SYN免疫阳性产物的平均OD值明显减少,差异具有显著性(P 0.05)。SYN与PirB的免疫荧光双重标记染色显示：海马CA3区苔藓纤维终端和锥体神经元胞体内SYN与PirB两者存在共定位。Western blot定量分析：LPS处理30天后,皮质、海马内SYN蛋白质的表达均明显减少,差异有显著性(P0.05)。 高尔基染色结果显示：与对照组相比,实验组大鼠海马锥体神经元基树突长度、树突棘密度均明显减少,差异具有显著性(P0.05)。 Morris水迷宫行为学测试结果：在训练的第四天,与对照组相比,实验组大鼠寻找平台潜伏期延长,差异具有显著性(P0.05)；在空间搜索实验中,对照组大鼠能依靠空间线索找到平台所在区域,其运动轨迹最多位于原平台象限,其次较多地在原平台象限相邻的左右两侧象限寻找,很少跨至对侧象限；实验组大鼠基本围绕池壁游泳,运动轨迹呈随机分布于各象限之中,较少游向原平台附近。实验组动物在原平台象限游泳停留时间占总时间的百分比明显减少,差异具有显著性(P0.01)。 结论LPS能够诱导大鼠皮质、海马星形胶质细胞活化和PirB蛋白表达上调,且部分活化的星形胶质细胞表达PirB,而突触囊泡素SYN蛋白表达下调,海马锥体神经元基树突长度及树突棘密度减少,学习记忆功能受损。海马CA3区苔藓纤维终末SYN阳性产物与PirB阳性产物共存。PirB可能参与脑内炎症突触可塑性改变和学习记忆功能缺失的免疫调节。
[Abstract]:Objective 1. to investigate the expression of PirB, the expression of synaptic vesicle and the change of animal behavior in chronic inflammatory brain neurons and astrocytes induced by lipopolysaccharide (LPS), and 2. to explore the immunological modulation of PirB in the loss of synapses and the loss of learning and memory function in chronic inflammatory neurons.
Methods the normal adult SD rats were randomly divided into experimental and control groups. LPS and PBS were injected into the right hippocampus of rats by stereotactic technique. The animals survived for 30 days. The expression of rat cerebral cortex, hippocampus PirB, synaptic vesicular vesicle and the expression of GFAP in astrocytes were detected by immunohistochemical method. The image analysis system was used to detect the expression of GFAP in the astrocytes. The number of positive cells and the intensity of staining, and quantitative analysis by Western blot. The changes in the structure of brain neurons in rats were observed by Golgi staining. The dual labeling technique of immunofluorescence was used to observe the coexistence of PirB positive cells and neurons, astrocytes and the relationship between the PirB positive cells and the synapses; the Morris water maze test was used. The learning and spatial memory abilities of rats in the experimental group were evaluated.
Results the GFAP positive astrocytes were distributed in the whole hippocampal structure, pyriform and olfactory cortex, and the white matter like callosum below the cortex. The hippocampal injection of PBS in the control group, the hippocampus and the GFAP positive astrocytes in the cortex showed the resting morphological characteristics: small cell body, long branching and pale stain. Intradypal injection in the hippocampus. The GFAP positive astrocytes in the hippocampus, pyriform and olfactory cortex of the experimental group of LPS were activated obviously, which showed that the cell body was obviously enlarged and the protuberance became hypertrophy. The quantitative analysis showed that the number of astrocytes and optical density of GFAP positive astrocytes in the hippocampus CA3 and dentate gyrus increased obviously after 30 days, and the difference was significant. P0.01.
The PirB positive neuron like cells in the V layer of the PBS injection group showed a round, oval and conical shape. The immune reaction products were brown and mainly on the cell membrane. After the injection of LPS30 in the hippocampus, the larger PirB positive neuron like cells and the smaller PirB positive glial like cells were mainly distributed in the cortex IV and the V layer. The pyramidal cell layer of the hippocampal CA1-CA3 region, the dentate gyrus granular cell layer, the immune reaction product located in the cytoplasm and the protuberance. Quantitative analysis showed that compared with the control group, the number and the OD value of the PirB positive neurons in the cortex and hippocampus were significantly increased after the treatment of 30d, and the difference was significant (P0.005).Western blot quantitative analysis: LPS treatment 30 days later, injection. In the lateral cortex, the expression of PirB protein in the hippocampus was significantly increased, the difference was significant (P0.001).PirB and MAP-2, GFAP, CDllb immunofluorescence double labeling staining showed that the cortex, the hippocampal PirB and MAP-2 positive neurons were Co located, PirB and GFAP positive astrocytes were partially Co located, experimental hippocampus hippocampus GFAP positive astrocytes were significantly activated, while PirB expression in activated astrocytes was up-regulated, but no double staining of PirB and CDllb positive staining was observed.
The SYN positive products were dot like brown particles, and the hippocampus of rats treated with PBS was mainly distributed in the beginning layer of the hippocampal CA1 region, the radiation layer and the molecular layer, the end of the almond angle nerve fibers in the CA3 region and the non cellular layer of the dentate gyrus. Compared with the control group, the average o value of the SYN immunoreactive products in the CA1 region of the hippocampus and the dentate gyrus treated with LPS was significantly reduced. Less significant (P 0.05).SYN and PirB immunofluorescence double labeling staining showed that there was a co localization of.Western blot in the hippocampal CA3 region moss fiber terminals and the SYN and PirB in the pyramidal neurons: 30 days after the LPS treatment, the expression of SYN protein in the cortex and hippocampus decreased significantly, and the difference was significant (P0.05).
The results of Golgi staining showed that compared with the control group, the length of the dendrites of the hippocampal pyramidal neurons and the density of the dendrites of the hippocampal neurons in the experimental group were significantly reduced, and the difference was significant (P0.05).
Morris water maze behavior test results: in the fourth day of training, compared with the control group, the incubation period of the experimental group was longer than the control group, the difference was significant (P0.05). In the space search experiment, the control group could rely on the spatial clue to find the area where the platform was located, and the track was most located in the original platform quadrant, followed by more. The left and right quadrants of the original platform quadrant were found and rarely crossed to the contralateral quadrant. The experimental group was basically swimming around the wall of the pool, and the motion trajectory was randomly distributed in the quadrants, and less in the vicinity of the original platform. The percentage of the total time of the experimental group in the original platform quadrant was significantly reduced and the difference was significant. Sex (P0.01).
Conclusion LPS can induce rat cortex, the activation of astrocytes and the expression of PirB protein in hippocampus, and some activated astrocytes express PirB, while the expression of synaptic vesicular SYN protein is down, the dendrite length and dendrite density of hippocampal pyramidal neurons are reduced, and the learning and memory function is impaired. SYN Yang of moss fiber terminals in hippocampus CA3 region is SYN positive. Coexistence of sex products with PirB positive products may involve.PirB in inflammatory synaptic plasticity and immune regulation in learning and memory deficits.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2136813
[Abstract]:Objective 1. to investigate the expression of PirB, the expression of synaptic vesicle and the change of animal behavior in chronic inflammatory brain neurons and astrocytes induced by lipopolysaccharide (LPS), and 2. to explore the immunological modulation of PirB in the loss of synapses and the loss of learning and memory function in chronic inflammatory neurons.
Methods the normal adult SD rats were randomly divided into experimental and control groups. LPS and PBS were injected into the right hippocampus of rats by stereotactic technique. The animals survived for 30 days. The expression of rat cerebral cortex, hippocampus PirB, synaptic vesicular vesicle and the expression of GFAP in astrocytes were detected by immunohistochemical method. The image analysis system was used to detect the expression of GFAP in the astrocytes. The number of positive cells and the intensity of staining, and quantitative analysis by Western blot. The changes in the structure of brain neurons in rats were observed by Golgi staining. The dual labeling technique of immunofluorescence was used to observe the coexistence of PirB positive cells and neurons, astrocytes and the relationship between the PirB positive cells and the synapses; the Morris water maze test was used. The learning and spatial memory abilities of rats in the experimental group were evaluated.
Results the GFAP positive astrocytes were distributed in the whole hippocampal structure, pyriform and olfactory cortex, and the white matter like callosum below the cortex. The hippocampal injection of PBS in the control group, the hippocampus and the GFAP positive astrocytes in the cortex showed the resting morphological characteristics: small cell body, long branching and pale stain. Intradypal injection in the hippocampus. The GFAP positive astrocytes in the hippocampus, pyriform and olfactory cortex of the experimental group of LPS were activated obviously, which showed that the cell body was obviously enlarged and the protuberance became hypertrophy. The quantitative analysis showed that the number of astrocytes and optical density of GFAP positive astrocytes in the hippocampus CA3 and dentate gyrus increased obviously after 30 days, and the difference was significant. P0.01.
The PirB positive neuron like cells in the V layer of the PBS injection group showed a round, oval and conical shape. The immune reaction products were brown and mainly on the cell membrane. After the injection of LPS30 in the hippocampus, the larger PirB positive neuron like cells and the smaller PirB positive glial like cells were mainly distributed in the cortex IV and the V layer. The pyramidal cell layer of the hippocampal CA1-CA3 region, the dentate gyrus granular cell layer, the immune reaction product located in the cytoplasm and the protuberance. Quantitative analysis showed that compared with the control group, the number and the OD value of the PirB positive neurons in the cortex and hippocampus were significantly increased after the treatment of 30d, and the difference was significant (P0.005).Western blot quantitative analysis: LPS treatment 30 days later, injection. In the lateral cortex, the expression of PirB protein in the hippocampus was significantly increased, the difference was significant (P0.001).PirB and MAP-2, GFAP, CDllb immunofluorescence double labeling staining showed that the cortex, the hippocampal PirB and MAP-2 positive neurons were Co located, PirB and GFAP positive astrocytes were partially Co located, experimental hippocampus hippocampus GFAP positive astrocytes were significantly activated, while PirB expression in activated astrocytes was up-regulated, but no double staining of PirB and CDllb positive staining was observed.
The SYN positive products were dot like brown particles, and the hippocampus of rats treated with PBS was mainly distributed in the beginning layer of the hippocampal CA1 region, the radiation layer and the molecular layer, the end of the almond angle nerve fibers in the CA3 region and the non cellular layer of the dentate gyrus. Compared with the control group, the average o value of the SYN immunoreactive products in the CA1 region of the hippocampus and the dentate gyrus treated with LPS was significantly reduced. Less significant (P 0.05).SYN and PirB immunofluorescence double labeling staining showed that there was a co localization of.Western blot in the hippocampal CA3 region moss fiber terminals and the SYN and PirB in the pyramidal neurons: 30 days after the LPS treatment, the expression of SYN protein in the cortex and hippocampus decreased significantly, and the difference was significant (P0.05).
The results of Golgi staining showed that compared with the control group, the length of the dendrites of the hippocampal pyramidal neurons and the density of the dendrites of the hippocampal neurons in the experimental group were significantly reduced, and the difference was significant (P0.05).
Morris water maze behavior test results: in the fourth day of training, compared with the control group, the incubation period of the experimental group was longer than the control group, the difference was significant (P0.05). In the space search experiment, the control group could rely on the spatial clue to find the area where the platform was located, and the track was most located in the original platform quadrant, followed by more. The left and right quadrants of the original platform quadrant were found and rarely crossed to the contralateral quadrant. The experimental group was basically swimming around the wall of the pool, and the motion trajectory was randomly distributed in the quadrants, and less in the vicinity of the original platform. The percentage of the total time of the experimental group in the original platform quadrant was significantly reduced and the difference was significant. Sex (P0.01).
Conclusion LPS can induce rat cortex, the activation of astrocytes and the expression of PirB protein in hippocampus, and some activated astrocytes express PirB, while the expression of synaptic vesicular SYN protein is down, the dendrite length and dendrite density of hippocampal pyramidal neurons are reduced, and the learning and memory function is impaired. SYN Yang of moss fiber terminals in hippocampus CA3 region is SYN positive. Coexistence of sex products with PirB positive products may involve.PirB in inflammatory synaptic plasticity and immune regulation in learning and memory deficits.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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