氯喹抑制体外培养星形胶质细胞的激活

目的研究氯喹对体外培养大鼠海马星形胶质细胞激活的抑制作用,为癫痫的治疗提供实验依据。方法分离新生SD大鼠海马,体外培养星形胶质细胞,经纯化鉴定后分为:对照组、戊四氮(PTZ)组、氯喹干预组(25、50和75 mg/L),经相应处理后,分别用MTT法、免疫荧光、Western blot测定星形胶质细胞数量及活性、星形胶质纤维酸性蛋白(GFAP)及CyclinD1的表达量。结果与对照组比较,PTZ可激活星形胶质细胞的增殖,使GFAP、CyclinD1的表达量增加(P<0.05);与PTZ组比较,氯喹阻滞了PTZ激活的星形胶质细胞的增殖(P<0.05);氯喹可抑制PTZ激活星形胶质细胞异常增加的GFAP的表达;氯喹可抑制PTZ激活星形胶质细胞的CyclinD1的表达量(P<0.05);与对照组相比,3种结果均显示75 mg/L氯喹对体外培养星形胶质细胞激活的抑制作用较强,并可维持其在正常范围。结论氯喹具有抑制PTZ激活体外培养星形胶质细胞的作用,其可能通过抑制星形胶质细胞的增殖来发挥抗癫痫作用。     

柴胡皂苷a抑制PTZ诱导的小鼠海马星形胶质细胞活化

目的:探讨柴胡皂苷a(SSa)对戊四氮(PTZ)诱导的小鼠海马星形胶质细胞活化的抑制作用。方法:分离培养小鼠海马星形胶质细胞,将细胞随机分为对照组、PTZ组、PTZ+0.625 mg/L SSa组和PTZ+1.25 mg/L SSa组。通过免疫荧光染色检测胶质细胞原纤维酸性蛋白(GFAP)的表达来鉴定细胞;用MTT检测评估细胞活力;用流式细胞术检测各组细胞的周期变化;ELISA法检测各组细胞中GFAP和间隙连接蛋白43(Cx43)的表达水平;流式细胞术和Hoechst 33258染色检测各组细胞的凋亡情况。结果:体外原代培养的星形胶质细胞贴壁生长,细胞突起明显。免疫荧光显示星形胶质细胞呈GFAP阳性表达。与对照组比较,PTZ组细胞活力和G_2/M期细胞百分比显著增加(P0.05),GFAP和Cx43的表达水平也显著上调(P0.05);与PTZ组比较,PTZ+0.625 mg/L SSa组和PTZ+1.25 mg/L SSa组细胞活力和G_2/M期细胞百分比均明显下降,GFAP和Cx43的表达水平也降低,但细胞凋亡水平显著增加(P0.05)。结论:SSa能够显著抑制PTZ诱导的海马星形胶质细胞活化,抑制细胞增殖并诱导凋亡。     

颞叶癫痫患者颞叶皮层和海马组织内多药耐药相关蛋白1和胶质原纤维酸性蛋白共表达

目的 观察颞叶癫痫病人多耐药基因1（MDR1）、胶质原纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）在颞叶和海马组织内的表达。方法 癫痫组样本来自12例颞叶癫痫病例的手术切除标本,对照组为4例非癫痫病的尸检脑组织。应用双重免疫荧光组织化学方法结合激光共聚焦显微镜技术显示MDR1、GFAP和NSE在颞叶和海马组织内的表达。结果 对照组颞叶皮质和海马齿状回内均可见到许多GFAP表达阳性的星形胶质细胞和NSE表达阳性的神经元,未见到表达MDR1的细胞。癫痫组颞叶皮质和海马齿状回内GFAP阳性星形胶质细胞高于对照组。颞叶皮层和海马组织内可见星形胶质细胞MDR1 和GFAP 共表达现象。结论 颞叶难治性癫痫可能与星形胶质细胞的多药耐药性有关。     

慢病毒介导的RNA干扰通过下调白细胞介素6抑制损伤后 星形胶质细胞中波形蛋白的表达

目的：探讨用慢病毒介导的RNAi方法抑制白细胞介素6（ IL-6）,观察其对损伤后星形胶质细胞中波形蛋 白（ Vim）表达的影响。方法：选取3 日龄SD大鼠的大脑皮质,体外培养星形胶质细胞,分为载体组和病毒组, 2 组均制备细胞划伤模型,采用qRT-PCR 检测Vim mRNA的表达,免疫印迹检测Vim蛋白表达,用免疫荧光检测 Vim的定位,MTT检测划伤模型中星形胶质细胞增殖率,免疫荧光检测GFAP用于观察划伤模型中星形胶质细胞 活性。结果：划伤细胞2 d 后,病毒组的Vim mRNA和蛋白表达量较载体组明显下降。Vim主要在星形胶质细胞 胞质中表达。病毒组中星形胶质细胞增殖情况和活性低于载体组。结论：用慢病毒介导的RNAi方法抑制炎症因 子IL-6,可以下调反应性胶质化细胞中Vim的表达,影响星形胶质细胞胶质化反应,可能抑制胶质瘢痕形成,对 神经细胞的再生具有积极作用。     

汉坦病毒感染乳大鼠大脑皮层星形胶质细胞

目的 建立汉坦病毒（HTNV）76-118株或汉城病毒（SEOV）L99株感染乳大鼠大脑皮层星形胶质细胞的体外培养体系,并检测其对汉坦病毒的易感性。 方法 通过免疫荧光显微技术、免疫印迹法、反转录聚合酶链反应检测星形胶质细胞对汉坦病毒的易感性,并通过反转录聚合酶链反应检测病毒S片段和GFAP基因表达情况。 结果 HTNV 或SEOV感染星形胶质细胞后病毒核衣壳蛋白（NP）和GFAP的表达增加,且GFAP和NP共定位,两者可能相互作用,同时病毒S片段和GFAP基因表达增加。 结论 HTNV或SEOV能有效感染和激活星形胶质细胞,GFAP可能调节血脑屏障结构或功能的完整性、病毒NP合成及病毒复制。     

亚低温对缺氧缺血新生大鼠海马星形胶质细胞增殖和凋亡的影响

目的观察亚低温对新生大鼠海马星形胶质细胞增殖和凋亡的影响,以探讨亚低温对缺氧缺血脑损伤保护作用的机制。方法①体外实验：取3日龄大鼠海马脑片,培养至第4天用氧糖剥夺法制备标本,于33℃（亚低温组）和37℃培养48h（常温组）;对照组脑片不进行氧糖剥夺处理,37℃培养7d。采用免疫荧光染色方法观察培养脑片星形胶质细胞的活化和增殖。②体内实验：取7日龄大鼠,分手术组和假手术组。手术组永久性结扎左侧颈总动脉,然后置于含8%O2＋92%N2中缺氧2h,制备缺氧缺血模型,分为手术亚低温亚组和手术常温亚组。假手术组仅分离左侧颈总动脉,不结扎,不予缺氧处理,分为假手术常温亚组和假手术亚低温亚组。各组于术后3和7d处死取材,采用免疫荧光染色方法检测海马星形胶质细胞的活化、增殖和凋亡。结果①体外实验：氧糖剥夺后3d常温组较对照组胶质纤维酸性蛋白（GFAP）阳性细胞数量明显增多;亚低温组与常温组比较,GFAP阳性细胞数量明显减少。②体内实验：术后3和7d亚低温组GFAP阳性细胞数量较常温组显著降低,较常温对照组和亚低温对照组显著增高。GFAP与caspase-3免疫荧光双标记结果显示,术后3d常温组海马区84.5%GFAP阳性细胞表达caspase-3,亚低温组仅32.3%GFAP阳性细胞表达caspase-3,差异有统计学意义。结论亚低温能减轻新生大鼠缺氧缺血后海马星形胶质细胞的活化增殖和凋亡。     

海马内注射LPS后诱导大鼠皮质星形胶质细胞活化和PirB的表达

目的:探讨海马内注射LPS(lipopolysaccharide,LPS)后,大鼠皮质神经元和星形胶质细胞PirB(paired-immunoglobulin-like receptor B)的表达变化。方法:成年SD大鼠,分为对照组和实验组,用免疫组织化学法检测大鼠脑皮质PirB的表达及星形胶质细胞GFAP的表达变化,免疫荧光双重标记技术,观察星形胶质细胞与PirB阳性细胞的共存。结果:PBS注射的对照组动物脑皮质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LPS 30 d后,PirB阳性神经元样细胞和PirB阳性神经胶质样细胞主要分布于皮质Ⅳ、Ⅴ层,免疫反应产物位于胞质和突起内;另外,可见大鼠梨状皮质、内嗅皮质内GFAP阳性星形胶质细胞明显被激活,表现为细胞胞体相对增大,突起变粗。PirB分别和MAP-2、GFAP、CD11b免疫荧光双重标记染色显示:PirB和MAP-2阳性神经元的胞体存在共定位;PirB与GFAP阳性染色存在部分共定位,但未见PirB与CD11b阳性染色双标细胞。结论:LPS能诱导大鼠皮质星形胶质细胞活化和PirB蛋白表达上调,而且部分活化的星形胶质细胞表达PirB,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。     

布洛芬对戊四氮点燃癫痫大鼠星形胶质细胞的影响

目的:探讨不同剂量布洛芬对戊四氮(PTZ)点燃癫痫大鼠的影响及其作用机制。方法:雄性SD大鼠60只,随机分为对照组、PTZ组和PTZ+布洛芬组(按布洛芬剂量分为4组),分别对其进行干预,观察记录各组大鼠行为学及脑电图变化,同时观测布洛芬的不良反应,采用免疫荧光染色及Western Blot检测GFAP的表达情况。结果:PTZ组与对照组相比,痫样发作和星形胶质细胞增生明显(P 0. 05); PTZ+布洛芬各组痫样发作和星形胶质细胞增生情况较PTZ组降低,且剂量越高抑制作用越明显(P 0. 05),但不良反应的发生也越多。结论:布洛芬可通过抑制星形胶质细胞增生影响癫痫发作,且剂量越高抑制作用越强,但其不良反应也随剂量的增加而增加。     

体外炎性诱导星形胶质细胞激活及功能分析

目的 观察体外培养的星形胶质细胞在炎性刺激下的激活及功能变化。方法 体外培养C57BL/6小鼠皮层的星形胶质细胞,应用免疫细胞化学和Western blot等方法,测定炎性刺激下各组细胞形态学及促炎因子（iNOS、IL-6、TNF-α）和抗炎因子（ARG1、IL-10、TGF-β1）表达水平的变化情况。结果炎性刺激后,免疫荧光细胞染色结果显示,M1(LPS+IFN-γ)组细胞被异常激活,胞体出现肿胀变大、扭曲等形态改变,且GFAP阳性细胞数量明显增多（P<0.001 vs. control））。CCK-8细胞活力检测结果显示,LPS组、IFN-γ组及M1组的吸光度均升高,其中M1组显著升高（P<0.001 vs. control）。Western blot结果表明,M1组促炎因子（iNOS、IL-6和TNF-α）表达均明显升高（P<0.001 vs. control）;各组抗炎因子（ARG1、IL-10和TGF-β1）的表达也显著升高（P<0.01 or 0.001 vs. control）。结论 体外炎性刺激异常激活星形胶质细胞,形态和功能显著变化,且A1样星...     

小胶质细胞源性因子对培养星形胶质细胞的活化作用研究

目的和方法：本实验采用胶质细胞分离纯化培养、胶质原纤维酸性蛋白（glial fibric acidic protein,GFAP）表达的免疫荧光流式细胞仪检测及[³H]-TdR掺入等方法,观察离体条件下小胶质细胞条件培养液对星形胶质细胞增殖与GFAP表达的影响。结果：小胶质细胞条件培养液能明显促进培养星形胶质细胞的增殖和GFAP的表达。结论：小胶质细胞源性因子能刺激星形胶质细胞的活化,由此推测在体条件下小胶质细胞源性因子可能是引起反应性胶质化的因素之一。     

栓塞大鼠大脑中动脉对海马结构内星形胶质细胞的形态学影响

目的:探讨栓塞大鼠大脑中动脉是否可以引致海马结构内星形胶质细胞的形态学改变。方法:用免疫组织化学方法显示栓塞大脑中动脉大鼠、假手术大鼠和对照大鼠海马结构内的GFAP阳性的星形胶质细胞的形态和数量以及GFAP表达,对比组间差异,寻找梗塞对远离缺血中心区的海马结构内星形胶质细胞的影响。结果:栓塞大鼠大脑中动脉可以引致远离缺血中心区的海马结构内星形胶质细胞数量增多、体积增大和GFAP表达增加。结论:脑缺血损伤引起的胶质细胞激活不仅限于缺血中心区和边缘区,远离缺血中心区的脑区也可以出现神经胶质细胞形态学改变,即胶质细胞激活现象。     

The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes

We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell-cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.     

Curcumin attenuates the kainic acid-induced hippocampal cell death in the mice

Kainic acid (KA) induced oxidative stress is associated with hippocampal cell death. Recent studies suggest that curcumin, a potent antioxidant, may provide protection for KA-induced oxidative stress. We investigated the effects of curcumin treatment on hippocampal reactive astrocytes in mice with KA-induced seizures. Eighteen hours after curcumin treatment, mice were treated with KA (30 mg/kg, i.p.), and then sacrificed after a further 48 h. Using cresyl violet staining and TUNEL analysis, histological evaluation revealed cell death in the KA-treated hippocampus. However, marked cell death was not observed in mice treated with curcumin. In addition, curcumin treatment reduced the KA-induced immunoreactivity of caspase-3. Similarly, immunoreactivity analyses indicated that KA causes upregulation of hippocampal GFAP, eNOS, and HO-1 levels, all of which were reduced in animals those received the curcumin treatment. Our findings indicate that curcumin is a potent inhibitor of reactive astrocyte expression and thus, prevents hippocampal cell death. These results also support its potential for use in the treatment of neurodegenerative diseases.     

Structural characteristics of astrocytes from the rat hippocampus in postischemia

The aim of this investigation was to study the distribution and structural organization of rat hippocampal astrocytes containing immunoreactive glial fibrillary acidic protein (GFAP) after ischemic damage of the brain in the animals treated with intraventricular infusion of creatine as a neuroprotective drug, and in those which received no treatment. Using the methods of light microscopy and immunocytochemistry, the brain of 26 mature Sprague-Dawley (Koltushi) rats was studied. Some animals were narcotized and subjected to general brain ischemia (lasting for 12 min) followed by a reperfusion (for 7 days). Creatine was infused intraventricularly to 11 animals using an automatic Alzet osmotic minipump. It was found that GFAP-immunoreactive hippocampal astrocytes were concentrated within two major areas (stratum lacunosum-moleculare CA1 and fascia dentata stratum polymorphae). As a result of neuroprotective effect of creatine, moderate ischemic damage of the hippocampus was not followed by the changes in the zones of activated astrocyte localization. Redistribution of GFAP-positive astrocytes in postischemic period was caused by the loss of pyramidal neurons in cytoarchitectonic field CA1. Complete loss of pyramidal neurons in this hippocampal area resulted in a qualitatively new level of astrocyte activation--their proliferation.     

地塞米松和苯妥英钠对癫痫大鼠脑内的GFAP和Fos表达的抑制作用

目的：探讨糖皮质激素（GC）的抗痫效应和抗痫机制。方法：动物行为学观察和免疫细胞化学染色。结果：戊四氮（PTZ）可诱发癫痫大发作,如诺在注入PTZ前30min先注入地塞米松或苯妥英钠（DPH）能减轻或抑制大鼠癫痫发作症状。免疫细胞化学染色结果表明,PTZ致痫组大鼠大脑皮质、海马回、齿状有大量肥大的胶质原纤维酸性蛋白（GFAP）阳性的星形胶质细胞。GC或DPH抗痫组GFAP免疫反应明显减弱,阳性细胞数量减少,突起短而少,Fos蛋白在PTZ组大鼠致痫后1－1．5h有大量表达,而在上述两抗痫组显著少于PTZ致痫组。结论：1．通过与苯妥英钠（传统抗痫药）的抗痫效果相比较,进一步证明糖皮质激素具有抗痫效应。2．糖皮质激素的抗痫机制可能与抑制星形胶质细胞的活动有关。3．Fos蛋白表达的变化与癫痫活动有直接关系。     

脑缺血再灌流后海马区胶质纤维酸性蛋白的动态表达及意义

本研究目的在于 :观察脑缺血再灌流后海马区胶质纤维酸性蛋白的分布及动态表达 ,探讨其与缺血性神经元的联系。钳夹沙土鼠的双侧颈总动脉制造脑缺血模型 ,应用免疫荧光法染色。结果显示 :脑缺血再灌流后胶质纤维酸性蛋白的阳性反应主要分布于海马本部的始层、放射层、分子层及齿状回门区。再灌流 3 d,胶质纤维酸性蛋白反应增强 ;7～ 15 d,胶质纤维酸性蛋白反应达高峰 ;脑缺血再灌流 40 d和对照组相比胶质纤维酸性蛋白阳性反应仍维持较高水平。再灌流 3 0～ 40 d,CA1区锥体层胶质纤维酸性蛋白阳性细胞明显增强。本研究结果表明 :脑缺血再灌流后海马区星形胶质细胞活化及胶质纤维酸性蛋白表达增强长期保持在较高水平 ,星形胶质细胞的活化、增生可作为神经元受损可靠而敏感的指标     

Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus.

The developmental pattern of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes was investigated in the hippocampus (subfields CA1, CA3 and CA4) and in the dentate gyrus of male and female rats aged 11, 16, 30, 90 and 150 days by immunohistochemistry associated with image analysis. Analysis was centred on stratum radiatum, a hippocampal area rich in GFAP-immunoreactive astrocytes. The volume of different portions of hippocampus, the number and the size of astrocytes, the intensity of cell body GFAP immunostaining as well as the extension of astrocyte were assessed. A maturation pattern consisting in higher cellular expression of GFAP, an increase in overall cell size and expanding arborisation from the 11th to the 30th postnatal day, followed by stabilisation of these parameters until the 90th day of life, and a subsequent decrease in the oldest age group studied was found. A sex-related different temporal pattern of astrocytes maturation in size and GFAP content was observed in the CA1 subfield only. The increase of GFAP content during pre-weaning ages was less pronounced in females than in males as well as the decrease between the 90th and the 150th day of age. Moreover, the size of astrocytes was larger in females than in males at the 11th and 150th days of life. These findings suggest that hippocampal astrocytes undergo rapid maturation in the 1st month of postnatal life, followed by a slow consolidation of this process until the 3rd month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner probably as a response to the increased volume of hippocampus noticeable at this age.     

银杏叶提取物对局灶性脑缺血再灌注后GFAP表达的影响

目的研究银杏叶提取物(extract of Ginkgo biloba,EGB)对局灶性脑缺血再灌注后星形胶质细胞胶质纤维酸性蛋白(GFAP)表达的影响。方法大脑中动脉插线法制作大鼠局灶性脑缺血再灌注模型。75只Wistar大鼠随机分为假手术组、缺血再灌注组、EGB治疗组。缺血2h再灌注48h后,采用免疫组织化学法检测脑组织内GFAP蛋白的表达。结果缺血再灌注后可诱导脑组织GFAP表达增强,EGB可抑制缺血再灌注后GFAP的表达(P<0.05)。结论局灶性脑缺血再灌注后,可诱导脑组织GFAP表达增强,EGB可抑制脑缺血后星形胶质细胞GFAP的高表达,提示EGB可能对缺血诱导的星形胶质细胞活化具有抑制作用,这可能是EGB抗脑缺血损伤保护神经元作用的机制之一。