四环素调控系统修饰永生化大鼠星形胶质细胞株的构建及鉴定

目的构建四环素及其衍生物强力霉素诱导表达目的基因的永生化大鼠星形胶质细胞株。方法用脂质体法将逆转录病毒载体四环素调控系统中的质粒pRevTet-On转染病毒包装细胞 PT67,经筛选培养后获得病毒载体RevTet-On,采用RT-PCR进行鉴定,并应用NIH313细胞测定病毒滴度。将RevTet-On感染永生化大鼠星形胶质细胞,用有限稀释法挑选阳性细胞单克隆后扩大培养,每个克隆瞬时转染含萤光素酶报告基因的质粒pRevTRE-Lue,加入强力霉素48h后分别检测其萤光素酶活性值,挑选出强力霉素诱导表达高、背景表达低的细胞株并检测其诱导表达的时效、量效关系。结果 RT-PCR结果显示逆转录病毒包装成功,病毒最高滴度为7．4×105CFU／ml。RevTet-On转染永生化大鼠星形胶质细胞后挑选出48个单克隆,瞬时转染pRevTER-Luc后筛选出1株高表达低背景细胞株, 其诱导表达值为876．1 RLU,背景表达值为42．5 RLU,诱导倍数为20．6。该细胞株在加入诱导因子强力霉素1 h后目的基因即开始表达,在24 h时达到高峰,在强力霉素浓度100-2 000 ng/ml的范围内, 其诱导表达活性与药物浓度呈剂量依赖性。结论含四环素调控系统的永生化大鼠星形胶质细胞株诱导活性可靠,可用于调控表达外源基因的研究。     

Nestin expression persists in astrocytes of organotypic slice cultures from rat cortex

Nestin is an intermediate filament protein typical for neural precursor cells that is down-regulated in the post-natal rodent brain. Re-expression of nestin has been observed in reactive astrocytes after injury. In this study, organotypic slice cultures from rat cortex were examined for expression of nestin and glial fibrillary acidic protein between 2 and 8 weeks in culture. Immunoreactivity for nestin and glial fibrillary acidic protein was seen in astrocytes which persisted throughout the observation period. Immunofluorescence double labeling showed widespread co-localization of nestin and glial fibrillary acidic protein. Image analysis revealed that levels of nestin-immunoreactivity plateaued after 5 weeks in culture. By comparison nestin immunoreactivity was absent from glial cells of the cortex in mature rats. These immunohistochemical findings of a persistent expression of nestin in glial cells of organotypic slice culture of the rat cortex indicate a different time course of glial maturation in vitro. This difference could be related to the altered trophic stimulation in vitro; differences in neuronal maturation, activity or survival; slow degeneration of the vasculature; or intrinsic properties of astrocytes.     

Production of nitrite by primary rat astrocytes in response to pneumococci

Recent studies using a rat model of pneumococcal meningitis have shown that nitric oxide synthase (NOS) inhibitors greatly attenuated microvascular changes and brain edema formation. The site of NO production during bacterial meningitis is unknown. In this study we tested whether primary astrocyte cultures from neonatal rat cortex can be induced to release NO upon stimulation with pneumococci. NO production was assessed by measuring nitrite in the cell culture supernatant using the Griess reaction. Stimulation with heat-killed unencapsulated pneumococci (HKP) increased nitrite concentrations in astrocyte culture supernatants in a dose-dependent fashion. Administration of AT-nitro-L-arginine (L-NA), aminoguanidine, L-canavanine, cycloheximide, and dexamethasone prevented the increase in nitrite concentrations. Addition of L-arginine, but not of o-arginine, partially reversed the inhibitory effect of L-NA. Administration of SOD increased nitrite accumulation. Moreover, at 72 h after stimulation with heat-killed pneumococci (10⁷ cfu/ml) astrocytes showed an inducible NOS-like immunoreactivity. Accumulation of nitrite was also observed when rat cerebellar neurons and microglia were stimulated with HKP, whereas there was only a slight increase of nitrite in media of rat C6 glioma cells, but no increase of nitrite when the human glioblastoma cell line LN-229 was stimulated with HKP. There was a stronger increase in nitrite levels when astrocytes from Lewis rats were used compared to that from Wistar rats. In conclusion, our study indicates that astrocytes, neurons and microglia are inducible for NO production upon stimulation with pneumococci.     

工频磁场对星形胶质细胞间隙连接通讯功能的影响

目的　探讨工频磁场 (PFMF)是否有促癌或协同促癌作用。方法　利用荧光光漂白后再恢复法观察漂白细胞荧光强度的恢复以判断经间隙连接的细胞间通讯 ,以相对荧光强度恢复速率(CFIRR)作为对细胞间隙连接通讯 (GJIC)作用的评价指标 ,研究不同磁场强度单独作用或协同佛波酯(TPA)对星形胶质细胞GJIC功能的影响。结果　 3ng/mlTPA作用 1h时CFIRR的中位数 (Md)值为4 .53 % /min ,空白对照组为 9.74% /min ,两组的差异有显著性 (H =1 2 .0 84,P <0 .0 0 5)。 0 .8或 1 .6mT磁场作用 2 4h时CFIRR的Md 分别 8.2 5、6 .68% /min ,与空白对照组比较 ,差异无显著性 (H =32 .61 7,P >0 .0 5)。 0 .8或 1 .6mT磁场作用 2 3h ,再与TPA共同作用 1h时CFIRR的Md 分别为 3 .32、2 .85% /min ,与TPA组比较 ,差异无显著性 (H =2 .589,P >0 .0 5)。结论　 0～ 1 .6mT的 50Hz磁场单独作用不能抑制星形胶质细胞GJIC功能 ;协同TPA ,不能增强TPA对星形胶质细胞GJIC的抑制作用 ;但是磁场对星形胶质细胞GJIC的抑制作用随着磁场强度增强呈递增趋势     

凋亡敏感基因在短暂性缺氧再复氧复注血清后的大鼠星形胶质细胞中的表达及其意义

目的研究一种新发现的抗氧化蛋白质--凋亡敏感基因(SAG)在短暂性缺氧再复氧复注血清诱导的细胞坏死和凋亡中的作用.方法用短暂性缺氧再复氧复注血清来诱导原代培养的大鼠大脑皮质星形胶质细胞损伤,用免疫细胞化学方法检测凋亡敏感基因的表达,并作图像分析;用流式细胞仪检测胶质细胞在短暂缺氧再复氧复注血清后不同时间点的凋亡率.结果凋亡敏感基因在缺氧15 min再复氧复注血清5 h后表达最高,在复氧复注血清16 h后恢复至对照组水平;细胞凋亡率在缺氧15 min再复氧复注血清1h时达到最高,而在再复氧复注血清5 h后降至对照组水平.结论凋亡敏感基因具有抗凋亡的作用,可减轻星形胶质细胞的缺血再灌注损伤.     

Astrocyte RNA in relation to neuronal RNA depletion in Alzheimer's disease

Summary A new double-staining procedure, in which the techniques of immunocytochemistry of glial fibrillary acidic protein (GFAP) and quantitative microdensitometry of azure B-RNA were combined, was used to study nucleic acid alterations in fibrous astrocytes in Alzheimer's disease (AD). RNA contents of GFAP-positive cells of the hippocampal endplate (Rose's H₃-H₅ fields) and the dentate gyrus molecular layer were determined in ten autopsy-proven AD patients (ages 51–88) and ten age-matched, non-demented control. In addition, RNA contents of pyramidal neurons of the endplate were examined. While there were no differences in RNA contents of astrocytes of either region between AD patients and controls, neuronal RNA was markedly depleted. These data suggest that astrocytes maintain protein synthetic capabilities in AD and that RNA loss is limited to the neuronal compartment.Supported by Grants 1P01-AG05119 and 1P50-AG05144 from the National Institutes of Health and by a Small Research Project Award from the University of Kentucky Medical Center     

Virulent and attenuated canine distemper virus infects multiple dog brain cell types in vitro.

Canine Distemper Virus (CDV) produces an encephalitis in dogs that varies with viral strain. We have studied the cell tropisms of two virulent strains (CDV-SH and CDV A75-17) and an attenuated strain, Rockborn (CDV-RO), in cultured canine brain cells. Infected cell types were identified by double immunofluorescent labeling of specific cell markers and viral antigens. All viral strains studied produced infection in astrocytes, fibroblasts, and macrophages. Neurons were not infected by CDV A75-17 but were rapidly infected by CDV-SH and CDV-RO. Multipolar oligodendrocytes were very rarely infected by any of the virus strains. In contrast, a morphologically distinct subset of bipolar oligodendrocytes were commonly infected by CDV-SH and CDV-RO. The kinetics of infection in the astrocytes, oligodendrocytes, neurons, and macrophages varied between strains. Both CDV-SH and CDV-RO rapidly infected bipolar oligodendrocytes, astrocytes, neurons, and macrophages by 14 days post infection while infection by CDV A75-17 was delayed until after 28-35 days post infection. The differences in the growth kinetics and cell tropisms for some brain cells, exhibited by the three viral strains examined in this in vitro study, may relate to the different CNS symptoms that these strains produce in vivo.     

Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM

RHAMM (Receptor for Hyaluronic Acid Mediated Motility) has been identified as a receptor for the extracellular matrix component hyaluronan (HA) and was recently shown to be essential for the locomotion of normal and transformed peripheral cells. Until now the potential role of RHAMM in the motility of neural-derived cells has not been investigated. Here, we report that cultured primary astrocytes, astrocyte cell lines, and microglia express this receptor and exhibit RHAMM-dependent motility. Immunocytochemical localization of RHAMM showed that it was often present as aggregates at the periphery of cells in contact with one another or concentrated on protruding processes of isolated cells. Glial cells contained 50 and 72 kDa forms of RHAMM, and both of these forms were found to have HA binding capacity. Time lapse imaging of cell locomotion revealed a significant inhibition of motility and process elongation by neutralizing anti-RHAMM antibodies and by peptides corresponding to the HA binding domains of RHAMM. These results demonstrate that RHAMM serves a role in glial cell locomotion in vitro and provide the basis for investigations of the motile behavior of glial cells in vivo after CNS injury.     

Allogeneic bone marrow stromal cells promote glial–axonal remodeling without immunologic sensitization after stroke in rats

We evaluated the effects of allogeneic bone marrow stromal cell treatment of stroke on functional outcome, glial–axonal architecture, and immune reaction. Female Wistar rats were subjected to 2 h of middle cerebral artery occlusion. Rats were injected intravenously with PBS, male allogeneic ACI – or syngeneic Wistar –bone marrow stromal cells at 24 h after ischemia and sacrificed at 28 days. Significant functional recovery was found in both cell-treated groups compared to stroke rats that did not receive BMSCs, but no difference was detected between allogeneic and syngeneic cell-treated rats. No evidence of T cell priming or humoral antibody production to marrow stromal cells was found in recipient rats after treatment with allogeneic cells. Similar numbers of Y-chromosome⁺ cells were detected in the female rat brains in both groups. Significantly increased thickness of individual axons and myelin, and areas of the corpus callosum and the numbers of white matter bundles in the striatum were detected in the ischemic boundary zone of cell-treated rats compared to stroked rats. The areas of the contralateral corpus callosum significantly increased after cell treatment compared to normal rats. Processes of astrocytes remodeled from hypertrophic star-like to tadpole-like shape and oriented parallel to the ischemic regions after cell treatment. Axonal projections emanating from individual parenchymal neurons exhibited an overall orientation parallel to elongated radial processes of reactive astrocytes of the cell-treated rats. Allogeneic and syngeneic bone marrow stromal cell treatment after stroke in rats improved neurological recovery and enhanced reactive oligodendrocyte and astrocyte related axonal remodeling with no indication of immunologic sensitization in adult rat brain.     

RNA干扰抑制马桑内酯诱导的大鼠星形胶质细胞Mdr1基因的表达

目的观察RNA干扰对培养的大鼠星形胶质细胞逆转P-糖蛋白介导的多药耐药现象的影响。方法用短发夹RNA表达载体p-SIREN shuttle质粒转染马桑内酯诱导表达P-糖蛋白的星形胶质细胞，荧光定量聚合酶链反应（PCR）测定Mdr1 mRNA的表达量，免疫细胞化学方法检测P-糖蛋白表达的变化，流式细胞术检测转染前后细胞罗丹明蓄积外排的变化。结果建立表达P-糖蛋白的星形胶质细胞模型，有效地将p-SIREN shuttle质粒转染该细胞模型。转染后实验组细胞Mdr1 mRNA水平被抑制达67．70％，P-糖蛋白表达明显低于对照组（P〈0．01），实验组P-糖蛋白作用底物罗丹明的细胞外泵出率（23．08％）明显低于对照组（78．35％，P〈0．01）。结论RNA干扰对马桑内酯诱导的星形胶质细胞Mdr1表达有明显抑制作用，并且在很大程度上逆转多药耐药蛋白的耐药作用。