免疫相關(guān)受體PirB在慢性炎癥腦神經(jīng)元突觸丟失中的作用
發(fā)布時間：2018-07-21 20:48
【摘要】：目的1.探討內(nèi)毒素脂多糖誘導(dǎo)的慢性炎癥腦神經(jīng)元和星形膠質(zhì)細(xì)胞PirB的表達(dá)變化,突觸囊泡素的表達(dá)及動物行為學(xué)的改變;2.探討PirB在慢性炎癥腦神經(jīng)元突觸丟失和學(xué)習(xí)記憶功能缺失中的免疫調(diào)節(jié)作用。 方法正常成年SD大鼠,隨機(jī)分為實(shí)驗(yàn)組和對照組,依賴立體定位技術(shù),在大鼠右側(cè)海馬內(nèi)分別注射LPS和PBS,動物存活30天。用免疫組織化學(xué)法檢測大鼠腦皮質(zhì)、海馬PirB、突觸囊泡素的表達(dá)及星形膠質(zhì)細(xì)胞GFAP的表達(dá)變化,用圖像分析系統(tǒng)檢測陽性細(xì)胞數(shù)目和染色強(qiáng)度；并采用Western blot技術(shù)對其進(jìn)行定量分析。高爾基染色觀察大鼠腦神經(jīng)元結(jié)構(gòu)的改變。免疫熒光雙重標(biāo)記技術(shù),觀察PirB陽性細(xì)胞與神經(jīng)元、星型膠質(zhì)細(xì)胞的共存及PirB陽性細(xì)胞與突觸的關(guān)系;Morris水迷宮測試用于評估實(shí)驗(yàn)組大鼠的學(xué)習(xí)和空間記憶能力。 結(jié)果GFAP陽性星形膠質(zhì)細(xì)胞分布在整個海馬結(jié)構(gòu)、梨狀皮質(zhì)和內(nèi)嗅皮質(zhì)、皮質(zhì)以下的白質(zhì)如胼胝體等部位。海馬內(nèi)注射PBS的對照組動物,海馬、皮質(zhì)內(nèi)GFAP陽性的星形膠質(zhì)細(xì)胞展示出靜息的形態(tài)特征：胞體小,分支細(xì)長,染色較淡。海馬內(nèi)注射LPS的實(shí)驗(yàn)組動物海馬、梨狀皮質(zhì)和內(nèi)嗅皮質(zhì)內(nèi)GFAP陽性星形膠質(zhì)細(xì)胞明顯被激活,表現(xiàn)為細(xì)胞胞體明顯增大,突起變肥大；定量分析表明,LPS處理30天后,海馬CA3區(qū)和齒狀回內(nèi)GFAP陽性星形膠質(zhì)細(xì)胞數(shù)目和光密度OD值明顯增加,差異有顯著性(P0.01)。 PBS注射組動物腦皮質(zhì)V層內(nèi)可見PirB陽性神經(jīng)元樣細(xì)胞,呈圓形、卵圓形和錐體形,免疫反應(yīng)產(chǎn)物呈棕色,主要位于細(xì)胞膜上；海馬內(nèi)注射LPS30天后,可見較大的PirB陽性神經(jīng)元樣細(xì)胞和較小的PirB陽性神經(jīng)膠質(zhì)樣細(xì)胞主要分布于皮質(zhì)Ⅳ、V層、海馬CA1-CA3區(qū)錐體細(xì)胞層、齒狀回顆粒細(xì)胞層,免疫反應(yīng)產(chǎn)物位于胞質(zhì)和突起內(nèi)。定量分析表明,與對照組比較,LPS處理30d后,皮質(zhì)、海馬內(nèi)PirB陽性神經(jīng)元的數(shù)目和OD值明顯增加,差異有顯著性(P0.005)。Western blot定量分析：LPS處理30天后,注射側(cè)皮質(zhì)、海馬內(nèi)PirB蛋白質(zhì)的表達(dá)均明顯增加,差異有顯著性(P0.001)。PirB分別與MAP-2、GFAP、CDllb的免疫熒光雙重標(biāo)記染色顯示：皮質(zhì)、海馬內(nèi)PirB和MAP-2陽性神經(jīng)元的胞體存在共定位；PirB與GFAP陽性星形膠質(zhì)細(xì)胞存在部分共定位,實(shí)驗(yàn)組海馬內(nèi)GFAP陽性星形膠質(zhì)細(xì)胞明顯被激活,活化的星形膠質(zhì)細(xì)胞PirB表達(dá)上調(diào),但未見PirB與CDllb陽性染色雙標(biāo)細(xì)胞。 SYN陽性產(chǎn)物呈點(diǎn)狀棕色顆粒,在PBS處理的大鼠海馬主要分布于海馬CA1區(qū)的始層、輻射層及分子層,CA3區(qū)阿蒙氏角神經(jīng)纖維終末以及齒狀回的非細(xì)胞層。與對照組相比,LPS處理的大鼠腦海馬CA1區(qū)、齒狀回內(nèi)SYN免疫陽性產(chǎn)物的平均OD值明顯減少,差異具有顯著性(P 0.05)。SYN與PirB的免疫熒光雙重標(biāo)記染色顯示：海馬CA3區(qū)苔蘚纖維終端和錐體神經(jīng)元胞體內(nèi)SYN與PirB兩者存在共定位。Western blot定量分析：LPS處理30天后,皮質(zhì)、海馬內(nèi)SYN蛋白質(zhì)的表達(dá)均明顯減少,差異有顯著性(P0.05)。 高爾基染色結(jié)果顯示：與對照組相比,實(shí)驗(yàn)組大鼠海馬錐體神經(jīng)元基樹突長度、樹突棘密度均明顯減少,差異具有顯著性(P0.05)。 Morris水迷宮行為學(xué)測試結(jié)果：在訓(xùn)練的第四天,與對照組相比,實(shí)驗(yàn)組大鼠尋找平臺潛伏期延長,差異具有顯著性(P0.05)；在空間搜索實(shí)驗(yàn)中,對照組大鼠能依靠空間線索找到平臺所在區(qū)域,其運(yùn)動軌跡最多位于原平臺象限,其次較多地在原平臺象限相鄰的左右兩側(cè)象限尋找,很少跨至對側(cè)象限；實(shí)驗(yàn)組大鼠基本圍繞池壁游泳,運(yùn)動軌跡呈隨機(jī)分布于各象限之中,較少游向原平臺附近。實(shí)驗(yàn)組動物在原平臺象限游泳停留時間占總時間的百分比明顯減少,差異具有顯著性(P0.01)。 結(jié)論LPS能夠誘導(dǎo)大鼠皮質(zhì)、海馬星形膠質(zhì)細(xì)胞活化和PirB蛋白表達(dá)上調(diào),且部分活化的星形膠質(zhì)細(xì)胞表達(dá)PirB,而突觸囊泡素SYN蛋白表達(dá)下調(diào),海馬錐體神經(jīng)元基樹突長度及樹突棘密度減少,學(xué)習(xí)記憶功能受損。海馬CA3區(qū)苔蘚纖維終末SYN陽性產(chǎn)物與PirB陽性產(chǎn)物共存。PirB可能參與腦內(nèi)炎癥突觸可塑性改變和學(xué)習(xí)記憶功能缺失的免疫調(diào)節(jié)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
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