低氧诱导体外培养大鼠星形胶质细胞VEGF的表达

目的　血管内皮细胞生长因子 (VEGF)是一种高度特异性的促血管内皮细胞生长的因子 ,探讨其在低氧情况下缺血组织血管形成的影响。方法　用免疫组织化学法和RT RCR法检测了正常培养和低氧复氧诱导情况下体外培养大鼠星形胶质细胞中VEGF蛋白及VEGFmRNA表达。结果　正常培养的大鼠星形胶质细胞中有VEGF表达 ,但表达量较低 ,低氧复氧诱导后星形胶质细胞中VEGF表达量会增加 ,且随着复氧时间延长表达量增加 ,6h到达高峰 ,以后又逐渐降低。结论　低氧复氧可能诱导大鼠星形胶质细胞代偿性增生 ,使VEGFmRNA表达上调而促进血管再生 ,这在一定程度上可减少缺血对神经元的影响。     

TNFα刺激的星形胶质细胞条件培养液对海马神经元的兴奋作用

目的:探讨肿瘤坏死因子-α刺激的星形胶质细胞对神经元的作用。材料和方法:体外纯化培养大鼠海马星形胶质细胞,采用反相高效液相法测定TNFα刺激后细胞培养液内谷氨酸的含量;将TNFα刺激后的星形胶质细胞条件培养液（Astrocytic Conditioned Medium,ACM）作用于培养的海马神经元,运用免疫细胞化学方法和图像分析技术研究神经元NF-κBp65和谷氨酸的表达。结果:（1）TNFα可明显促进星形胶质细胞释放谷氨酸;（2）ACM作用15min即可诱导神经元NF-κBp65的核表达,30min达高峰,180min恢复至对照水平;（3）ACM作用60min可使谷氨酸免疫反应阳性神经元平均光密度明显升高,持续至240min。结论:TNFα刺激的星形胶质细胞可通过释放谷氨酸等可溶性物质使神经元快速激活、兴奋性升高。     

星形胶质细胞转分化为神经元在神经再生中的研究进展

<正>星形胶质细胞是广泛存在于中枢神经系统内的一类胶质细胞,在建立和维持正常脑功能以及脑损伤后修复中具有重要作用。近几年发现,特定区域脑损伤后星形胶质细胞发生活化,甚至转分化为神经元谱系,表现出类似于神经前体细胞的特性,这一重要的潜能使得星形胶质细胞成为神经再生领域研究的焦点。本文就星形胶质细胞的基本特性、其转分化为神经元的模型以及介导转分化发生的细胞与分子学     

桩蛋白在两型星形胶质细胞及神经元中的差异表达

目的:观察桩蛋白在大鼠两型星形胶质细胞及神经元中的差异表达情况。方法:通过细胞培养并结合免疫荧光和RT-PCR方法,观察了桩蛋白的mRNA及蛋白在大鼠两型星形胶质细胞T1A和T2A以及神经元中的表达。结果:RT-PCR显示体外培养的T1A表达桩蛋白mRNA,而T2A和神经元未表达桩蛋白mRNA;免疫荧光染色显示桩蛋白主要分布于T1A的胞浆及突起中。结论:桩蛋白在T1A中表达,但未表达于T2A和神经元中;本实验结果为进一步研究桩蛋白在T1A中的生物学作用奠定了基础。     

大鼠脑缺血再灌流时神经元损伤与星形胶质细胞的反应

为探讨星形胶质细胞在缺血性神经元损伤中的作用及其与神经元损伤的关系 ,本实验阻塞大鼠大脑中动脉 2 h,再灌流0 .5～ 48h建立短暂局灶性脑缺血模型 ,进行 H-E染色 ;通过胶质原纤维酸性蛋白和细胞核增殖抗原免疫组化单重或双重反应 ,TU NEL和胶质原纤维酸性蛋白免疫组化双重反应观察了神经元和星形胶质细胞的反应。结果表明 :再灌流 2 4h缺血区面积最大 ,再灌流 6h开始出现神经元不可逆变性 ,2 4h梗塞成熟 ;星形胶质细胞表现为反应性、营养不良性和退形变三种不同的形态特点。再灌流 48h时星形胶质细胞数量开始增多。 48h之内星形胶质细胞无增生 ,且有少量星形胶质细胞凋亡。这些结果提示脑缺血时星形胶质细胞反应与神经元损伤密切相关 ,反应性星形胶质细胞是其积极应答神经元损伤的结果 ,在维持神经元存活中起作用。     

星形胶质细胞条件培养液对缺氧损伤神经元的保护作用

为了探讨星形胶质细胞条件培养液(ACM)对缺氧损伤神经元的保护作用,本实验将新生的KM小鼠的皮层星形胶质细胞分离、纯化并传代培养第三代第5d后,将星形胶质细胞(Ast)条件培养液,加入到缺氧损伤的神经元细胞培养液中,观测其对缺氧神经元的存活及其合成和释放一氧化氮(NO)、乳酸脱氢酶(LDH)和胞膜ATP酶(ATPase)的影响。结果显示:10%和20%ACM对神经元存活有明显促进作用;与对照组相比,20%ACM还有效地减少了缺氧神经元NO、LDH的生成和分泌,保护和提高了ATPase的活性。以上结果提示ACM促进缺氧神经元的存活,其机制可能与减少NO、LDH合成释放及保护细胞膜上ATPase的活性有关。     

低氧刺激诱导体外培养的大鼠脑皮质星形胶质细胞内neuregulin-1表达上调

Neuregulin-1(NRG-1)是神经胶质及神经元产生的细胞间信号转导蛋白。该蛋白通过与ErbB受体结合,对神经系统的正常发育、成熟发挥重要作用,也在缺血性脑损伤时起神经保护作用。为探讨体外培养的星形胶质细胞受缺氧刺激后NRG-1的表达特点,本实验利用体外培养的大鼠脑皮质星形胶质细胞,采用免疫组织化学和Westernblot方法,比较了正常培养和低氧复氧条件下星形胶质细胞中NRG1的表达。结果显示:正常培养的星形胶质细胞中有NRG-1的表达,但含量不高;低氧复氧培养后星形胶质细胞NRG1的表达量随着复氧时间的延长缓慢增加,至复氧8h,其表达量陡然升高,达到峰值后又逐渐降低,甚至降至正常水平以下。本研究表明,在低氧缺血性脑损伤后,星形胶质细胞反应性大量产生NRG1的时间相对滞后。由此提示,低氧缺血性脑损伤后,立即使用外源性神经保护剂为宜。     

星形胶质细胞在体内直接重编程为神经元的研究进展

人类神经元几乎不具有再生能力,这也意味着神经元一旦受损或退化,神经系统的自我修复将无计可施。研究发现,星形胶质细胞作为一种广泛分布于中枢神经系统(CNS)中的神经胶质细胞,具备诱导转化为神经元的潜力。事实上,各国研究学者已经成功利用多种技术手段将来源不同的星形胶质细胞重编程为神经元。本文综述了脊髓损伤(SCI)、脑卒中(CS)、帕金森病(PD)、阿尔茨海默病(AD)和亨廷顿病(HD)、肌萎缩性脊髓侧索硬化症(ALS)等神经系统相关疾病中星形胶质细胞直接重编程为功能性神经元的再生策略。     

渗透压变化对缝隙连接蛋白在培养的大鼠下丘脑星形胶质细胞和神经元表达的影响

目的观察渗透压改变对培养的下丘脑星形胶质细胞的缝隙连接蛋白Connexin43（Cx43）和神经元的缝隙连接蛋白Cx32表达的影响。方法体外培养孕14d或新生1d SD大鼠下丘脑的神经元和星形胶质细胞并进行纯化和鉴定。实验各分两组,第1组：细胞由等渗培养液移至高渗培养液（含9％NaCl）分别放置1min、3min、5min、10min和15min后将液体吸出常规固定;第2组：细胞先置于高渗培养液中15min后换成等渗培养液,分别在1min、3min、5min和10min后将等渗培养液吸出常规固定。两者均进行抗Cx43或抗Cx32的免疫荧光染色,Confoeal显微镜观察。结果第1组,Cx43在刺激1min后在星形胶质细胞表达比对照组有所增加,3min达到高峰,以后逐渐降低,15min恢复正常;而Cx32在刺激后1min的神经元中表达比对照组增加,5min达到高峰,以后降低。15min恢复正常。第2组同样为Cx43在星形胶质细胞刺激1min后表达增加,3min达到高峰,以后降低,10min恢复正常;神经元的Cx32表达在刺激后5min达到高峰,以后降低,10min恢复正常。两组Cx43表达的高峰期均早于Cx32。结论Cx43和Cx32可能参与星形胶质细胞与神经元渗透压的调节。     

川芎嗪对缺氧缺血性脑损伤大鼠海马星形胶质细胞的影响

观察川芎嗪(tetramethylpyrazine,TMP)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)大鼠海马星形胶质细胞的影响。将90只7日龄Wistar大鼠随机分为假手术组、HIBD组、TMP治疗组,各组分别在0h、6h、12h、24h、48h五个时间点取右脑,透射电镜下观察海马CA1区星形胶质细胞超微结构变化,免疫组织化学法检测胶质纤维酸性蛋白(glial fibrillary a-cidic protein,GFAP)表达的变化。结果显示,在电镜下HIBD损伤组星形胶质细胞肿胀,体积增大,核形状不规则,核质比增大,核仁明显,位于近核膜处,细胞器明显减少,线粒体肿胀,嵴紊乱,有缺失,可见粗面内质网池扩张;TMP组星形胶质细胞损伤轻,形态基本正常。HIBD后海马CA1区星形胶质细胞反应明显增强(P<0.01),川芎嗪治疗后可明显减轻星形胶质细胞的反应程度(P<0.01)。以上结果提示川芎嗪可通过减轻HIBD后星形胶质细胞的反应程度发挥保护和修复神经细胞的作用。     

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.     

低氧预适应对小鼠海马神经元中TSC1表达的影响

目的:在体视显微镜下分割ICR小鼠海马CA1区和CA3区,研究结节性硬化症因子1(TSC1)在小鼠海马低氧中的神经保护作用。方法:ICR小鼠分为对照组(control)、低氧对照组(hypoxia)及低氧预适应组(HPC),在体视显微镜下观察海马形态并分割CA1区和CA3区;采用real time RT-PCR和Western Blot的方法分别检测小鼠海马组织TSC1 mRNA和蛋白的表达;采用免疫荧光检测小鼠海马组织TSC1荧光强度。结果:大脑冠状切片清晰显示出海马CA1区、CA3区和DG区; CA1区TSC1 mRNA在低氧组降低而在低氧预适应组增高;Western Blot和组织免疫荧光显示:与对照组相比,低氧预适应组CA1区TSC1表达增加;而CA3区TSC1在低氧组和低氧预适应组均增加。结论:TSC1的差异性表达可能提示TSC1可能参与了低氧预适应对低氧敏感的CA1区神经细胞的保护。     

大鼠GLUT3启动子的克隆及其缺糖调控活性的测定

目的: 构建葡萄糖转运体3(GLUT3)启动子的报告基因,并在正常和缺糖情况下检测其转录活性。方法: 使用生物信息学软件预测了GLUT3 的启动子序列,长度1 292 bp, 包含第1个外显子, 通过PCR及双酶切方法,从大鼠全血基因组DNA中获得GLUT3 基因编码序列, 包含GLUT3 启动子序列,然后克隆到报告基因pGL3-Basic载体上,构建GLUT3 启动子的报告基因;用脂质体转染法将pGL3-GLUT3与胸腺嘧啶脱氧核苷激酶(pRL-TK)共转染入PC12细胞中,以pRL-TK载体作内参照,分别给予正常和缺糖培养,采用双萤光素酶报告系统评估GLUT3 启动子的活性。结果: 经PCR方法扩增出GLUT3 启动子序列,测序与GenBank序列一致;转染后检测显示,该pGL3-GLUT3明显具有转录活性,而且在缺糖24 h情况下其双萤光素酶活性有明显升高。结论: 成功构建出GLUT3 启动子报告基因,pGL3-GLUT3表现出很好的缺糖诱导活性,缺糖24 h是研究pGL3-GLUT3在缺糖情况下转录调控很有意义的时间点。     

Apamin,a selective blocker of SKCa channels,inhibits posthypoxic hyperexcitability but does not affect rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the effects of apamin, a selective blocker of SK_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. Application of apamin (50 nM) to the hippocampal slices during hypoxic episodes significantly abolished posthypoxic hyperexcitability induced by brief hypoxic episodes. However, in contrast to our previous results with iberiotoxin, a selective blocker of BK_Ca channels, apamin significantly enhanced the depressive effect of brief hypoxia on the PS amplitude during hypoxic episode and did not abolish the rapid hypoxic preconditioning in CA1 pyramidal neurons. Present results indicate that SK_Ca channels, along with previously implicated BK_Ca channels, play an important role in the development of posthypoxic hyperexcitability induced by brief hypoxic episodes in CA1 pyramidal neurons. However, SK_Ca channels, in contrast to the BK_Ca channels, are not involved in the rapid hypoxic preconditioning in CA1 hippocampal region in vitro.     

ADM血浆浓度的动态变化在HIE早期诊断中的临床研究

目的：通过对足月新生儿缺氧缺血性脑病（hypoxic ischemic encephalopathy,HIE）脐血浆中肾上腺髓质素（adrenomedullin,ADM）水平变化与血清神经元特异性烯醇化酶（Nenron specific enolase,NSE）动态变化及相关性研究,评价脐血ADM在HIE中的诊断价值。方法：使用放射免疫分析（RlA）测定30例正常新生儿和110例窒息足月儿的脐血在出生后24h、3d、7d ADM、NSE的动态变化。结果：①HIE组脐血ADM浓度为168.15pg/ml,较非HIE组的106.18pg/ml和对照组的103.65pg/ml明显增高,有显著性差异（P〈0.01）。②HIE组ADM浓度变化与HIE的轻、中、重程度呈正相关,与NSE浓度变化密切相关（r=0.786,P〈0.01）。HIE组出生后24h血浆ADM浓度明显增高,经治疗后,轻、中度组,第7d基本恢复正常对照组水平,而重度组仍维持较高浓度。③不同脐血ADM浓度为截断值绘制ROC曲线,最佳截断值为117.5pg/ml。ADM〉118.0pg/ml对HIE诊断的准确度为88.9%、特异性为83.3%、敏感度为91%。结论：脐血检测ADM浓度比NSE具有早期预测、快速方便、无创、更敏感的优点。脐血ADM测定在HIE早期诊断中有重要临床意义。     

Comparative roles of ATP-sensitive K channels and Ca-activated BK channels in posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the comparative effects of glibenclamide (GC), a selective blocker of K⁺_ATP channels, and iberiotoxin (IbTX), a selective blocker of BK⁺_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. In contrast to GC (10 μM), IbTX (10 nM) significantly abolished both posthypoxic hyperexcitability and rapid hypoxic preconditioning induced by brief hypoxic episodes. These effects of IbTX did not depend on its ability to reduce the hypoxia-induced decrease of population spike (PS) amplitude during hypoxic episodes since GC (10 μM), comparatively with IbTX (10 nM), significantly reduced the depressive effect of hypoxia on the PS amplitude during hypoxic episodes but did not abolish both posthypoxic hyperexcitability and rapid hypoxic preconditioning in CA1 pyramidal neurons. Our results indicated that BK⁺_Ca channels, in comparison with K⁺_ATP channels, play a more important role in such repeated brief hypoxia-induced forms of neuroplasticity in hippocampal CA1 pyramidal neurons as posthypoxic hyperexcitability and rapid hypoxic preconditioning.     

银杏叶提取物预处理对VD模型大鼠海马CA1区P-GP和GLT-1表达的影响

目的:探讨银杏叶提取物(EGB761)预处理对血管性痴呆(VD)模型大鼠海马CA1区P-糖蛋白(P-GP)及谷氨酸转运体1(GLT-1)的影响。方法:大鼠分为假手术组、模型组和EGB761预处理组。EGB761预处理组在造模前7 d给予EGB761生理盐水混悬液灌胃,假手术组和模型组给予等体积的生理盐水灌胃。采用重复夹闭双侧颈总动脉(CCA)同时腹腔注射硝普钠溶液方法复制VD大鼠模型。Morris水迷宫检测大鼠空间学习记忆能力,用神经颗粒素(Ng)/P-GP和胶质细胞酸性蛋白(GFAP)/GLT-1免疫荧光双标法,观察海马CA1区神经元P-GP和胶质细胞GLT-1表达水平的变化,Western Blot技术检测VD大鼠海马区P-GP和GLT-1蛋白的表达量。结果:(1)Morris水迷宫检测显示,与假手术组相比,模型组大鼠的逃逸潜伏期(Escape latency,EL)明显延长(P0.01),而EGB761预处理组大鼠EL明显缩短(P0.01)。(2)阳性细胞计数观察到,与假手术组相比,模型组海马CA1区Ng/P-GP的阳性细胞数明显减少,GFAP/GLT-1的阳性细胞数则显著增多(P0.01),EGB761预处理组海马CA1区Ng/P-GP和GFAP/GLT-1的阳性细胞数较模型组均明显增多(P0.01)。(3)积分光密度值测定观察到,模型组和EGB761预处理组的Ng/P-GP和GFAP/GLT-1阳性细胞的IOD值较假手术组均明显增大(P0.01);EGB761预处理组的Ng/P-GP和GFAP/GLT-1阳性细胞IOD值均显著高于模型组(P0.01)。(4)Western Blot分析结果显示,模型组和EGB761预处理组大鼠海马CA1区P-GP和GLT-1蛋白表达的灰度值均比假手术组增大(P0.01),EGB761预处理组大鼠海马CA1区P-GP和GLT-1蛋白表达的灰度值均显著大于模型组(P0.01)。结论:EGB761预处理可能通过上调VD模型大鼠海马CA1区P-GP和GLT-1的表达来预防VD的发生。     

Correlation between local monocarboxylate transporter 1 (MCT1) and glucose transporter 1 (GLUT1) densities in the adult rat brain

Monocarboxylate transporters type 1 (MCT1) facilitate the transport of monocarboxylates across cell membranes of the blood-brain barrier and brain parenchymal cells. The present study had two aims: (1) to determine the local distribution of MCT1 in the brain; and (2) to compare the local densities of MCT1 with the local densities of the main nutritional transporters, glucose transporter GLUT1. Using immunoautoradiography of cryosections from rat brain, 32 brain structures were analyzed. (1) A heterogenous distribution pattern of MCT1 densities was observed throughout the brain. Compared to brain homogenate (100%), MCT1 densities ranged from 43 to 164% in the brain structures investigated. Local GLUT1 densities showed a comparable range (35-145%). (2) A close correlation was found between local MCT1 and local GLUT1 densities. As local GLUT1 densities reflect local glucose metabolism in the brain, we conclude that local MCT1 densities are adjusted to local glucose metabolism and transport.     

Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons

The extracellular signal-regulated kinases (ERK) signalling cascade is a key pathway that mediates the NMDA receptor (NMDAR)-dependent neuronal plasticity and survival. However, it is not clear yet how NMDARs regulate ERK activity. Stimulation of the NMDARs induces a complex modification of ERK that includes both ERK activation and inactivation and depends on particular experimental conditions. Here we show that there exists a differential restriction in the regulation of ERK activity that depends on the pool of NMDAR that was activated. The synaptic pool of NMDARs activates ERK whereas the extrasynaptic pool does not; on the contrary, it triggers a signalling pathway that results in the inactivation of ERK. As a result, simultaneous activation of both extrasynaptic and synaptic NMDAR using bath application of NMDA or glutamate (a typical protocol explored in the majority of studies) produced ERK activation that depended on the concentration of agonists and was always significantly weaker than those mediated by synaptic NMDARs. Since the activation of the extrasynaptic NMDA is attributed mainly to global release of glutamate occurring at pathological conditions including hypoxic/ischaemic insults, traumas and epileptic brain damage, the reported differential regulation of ERK cascade by NMDARs provides a unique mechanism for an early identification of the physiological and/or pathophysiological consequences of NMDAR activation. The negative regulation of the ERK activity might be one of the first signalling events determining brain injury and constitutes a putative target of new pharmacological applications.     

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.