缺血缺氧对体外培养星形胶质细胞细胞周期和增殖的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期和增殖的影响。方法 用流式细胞仪检测缺血缺氧后不同时问点星形胶质细胞细胞周期变化，并用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)和增殖细胞核抗原(PCNA)的表达水平。结果 体外缺血缺氧损伤后星形胶质细胞S期较正常组明显增高，6h达高峰，而随后则呈下降趋势。PCNA阳性反应损伤后表达均增加，6h表达最高；在缺血缺氧早期，GFAP阳性染色增强，6h最高；缺血缺氧12h后GFAP阳性染色变弱。结论 缺血缺氧损伤后星形胶质细胞活化进入增殖期；PCNA参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的活化密切相关。     

星形胶质细胞体外缺血缺氧损伤后炎症模型的建立及应用

目的 建立SD大鼠星形胶质细胞缺血缺氧损伤后炎症模型,探讨亚低温对星形胶质细胞缺血缺氧及损伤后炎症反应的影响.方法 体外原代培养新生SD大鼠星形胶质细胞,免疫荧光染色检测胶质纤维酸性蛋白(GFAP)进行鉴定.实验分正常对照组(C组)、单纯缺氧组(H组)和缺氧+白细胞组(H+W组),以无糖DMEM培养基、5%CO2+95%N2混合气体培养4 h诱导细胞缺氧模型.H+W组加入1 mLSD大鼠外周血白细胞(1×106/mL),C组加入等量培养液.将3组细胞分别置入37℃、34℃、32℃、30 ℃ CO2孵箱中作用24 h,应用速率法和LIVE/DEAD染色分别检测3组细胞在不同温度下乳酸脱氢酶(LDH)释放率的变化和形态学变化.结果 缺氧4 h即可造成星形胶质细胞的损伤.37℃时C组、H组、H+W组细胞LDH释放率依次升高,差异有统计学意义(P<0.05);与37℃相比,亚低温状态(34℃、32℃)下H组、H+W组细胞LDH释放率均降低;但在30℃时,则有明显升高,与32℃相比差异具统计学意义(P<0.05).结论 亚低温状态可明显降低星形胶质细胞的缺血缺氧性损伤及炎症性损伤,其机制可能并非通过单纯的抑制代谢来实现的.     

脑白质低灌注损伤后星形胶质细胞缝隙连接蛋白43的表达变化

目的研究慢性脑白质缺血后星形胶质细胞和缝隙连接蛋白Connexin43(Cx43)的变化。方法原代培养星形胶质细胞,建立体外慢性缺氧模型;双侧颈总动脉狭窄法,建立慢性低灌注脑白质损伤小鼠模型;免疫荧光共染观察星形胶质细胞活化与Cx43表达。Western蛋白定量分析髓鞘相关指标髓鞘相关糖蛋白MAG,星形胶质细胞标记物GFAP和Cx43的表达。结果与对照组相比,细胞慢性缺氧7d后,星形胶质细胞明显增生活化,伴随Cx43表达水平明显上调。Western blot发现,在慢性脑白质缺血过程中,MAG的表达逐渐降低,GFAP持续增高,Cx43表达明显上调。免疫荧光共标记可见,星形胶质细胞中Cx43表达上调,主要分布于胼胝体中央区。结论慢性脑白质缺血损伤过程伴随星形胶质细胞Cx43表达增加,Cx43可能成为临床治疗血管性认知障碍的新靶点。     

PPAR-γ对大鼠脊髓损伤后神经功能影响的研究

目的观察PPAR-γ对大鼠脊髓损伤后GFAP(胶质纤维酸性蛋白)及新生星形胶质细胞表达的影响。方法 SD大鼠108只,分成损伤组、PPAR-γ激动剂及拮抗剂治疗组。损伤后观察BBB评分、GFAP及新生星形胶质细胞的表达。结果激动剂治疗组BBB(BBB运动功能评分)及GFAP表达较损伤组GFAP的表达在1~2 w时间点内表达增加(P0.05),拮抗剂治疗组GFAP的表达较脊髓损伤组GFAP的表达在1~4 w时间点内表达减少(P0.05)。激动剂组新生星形胶质细胞在1~2 w明显增加(P0.05),而拮抗剂治疗组表达明显减少(P0.05),有统计学差异。结论 PPAR-γ激活后促进GFAP及星形胶质细胞的表达,起到神经保护作用。     

星形胶质细胞AQP4蛋白在缺氧/复氧条件下表达变化的研究

目的观察缺氧/复氧条件下星形胶质细胞形态和AQP4蛋白的表达变化以及葛根素对其表达变化的影响,探讨脑缺血再灌注损伤与AQP4的关系以及葛根素的干预作用。方法原代培养星形胶质细胞,用5%CO2+95%N2混合气体造成缺氧,以LDH漏出率及MTT降解率作为细胞受损指标,应用Western blot技术检验星形胶质细胞缺氧/复氧各个时间点AQP4蛋白的表达变化及葛根素的干预效果。结果体外培养的星形胶质细胞在缺氧环境下损伤不明显,随着复氧时间的延长细胞损害加重。AQP4蛋白在缺氧时表达与正常对照组无明显差异,复氧后表达升高并随时间延长呈增高趋势(P0.05)。葛根素干预组AQP4蛋白表达丰度与缺氧/复氧组无明显差异(P0.05)。结论星形胶质细胞AQP4蛋白表达变化与细胞损伤有明显相关性,葛根素对星形胶质细胞损伤的保护作用不是通过改变AQP4的表达来实现的。     

缺血缺氧对体外培养星形胶质细胞细胞周期和周期相关蛋白的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期及细胞周期相关蛋白的影响。方法 用流式细胞仪及Brdu掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力；用荧光免疫细胞化学技术测定增殖细胞核抗原(PCNA)及细胞周期蛋白cyclin D1的表达水平。结果 体外缺血缺氧损伤后S期星形胶质细胞较正常组明显增加，6h达高峰，Brdu掺入法显示损伤后6h星形胶质细胞的增殖活力最高，而随后S期细胞数目及细胞增殖活力都呈下降趋势。PCNA阳性反应损伤后表达增加，6h表达最高，而cyclin D1的表达在损伤后逐渐增加，在24h时达高峰。结论 缺血缺氧损伤激活星形胶质细胞，使其进入新的细胞周期，出现细胞的增殖反应；PCNA及cyclin D1参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的增殖活化密切相关。     

Nrf2在短暂性缺血再灌注大鼠星形胶质细胞模型中的表达变化及其意义

目的 观察星形胶质细胞短暂性缺血再灌注损伤过程中胞质和胞核的NF-E2相关因子2(Nrf2)表达变化,分析其核转位情况与细胞氧化损伤水平的相关性.方法 用缺血缺氧0.5h后再复氧复注血清诱导原代培养的大鼠大脑皮质星形胶质细胞损伤,酶标仪检测各时间点细胞活性氧簇(ROS)和谷胱甘肽(GSH)水平,MTT比色法测定细胞存活率,Western blot检测胞质和胞核内Nrf2表达水平.结果 随着缺血再灌注时间的延长,细胞ROS逐渐增加,GSH含量和存活率逐渐下降(P<0.05),但再灌注8h与12h比较,细胞ROS、GSH和存活率无显著性差异(P>0.05);缺血再灌注早期(再灌注0.5h),胞浆Nrf2表达减少,胞核Nrf2增多(P<0.05),继续再灌注损伤,胞浆和胞核Nrf2均减少,再灌注8h,胞浆和胞核Nrf2表达不再下降(P>0.05).结论 Nrf2核转位与短暂性缺血再灌注损伤造成星形胶质细胞氧化应激损伤具有相关性.     

新生儿缺氧缺血性脑病临床与神经病理的相关性研究

目的观察新生儿缺氧缺血性脑病临床与神经病理之间的相关性。方法纳入2014-05—2015-04缺氧缺血性脑病患儿90例,进行免疫组化法观察不同生存时间的原纤维性酸性蛋白(GFAP)标记的星形胶质细胞的变化;比较宫内窘迫组(慢性缺氧缺血)、出生窒息组(急性缺氧缺血)、混合性缺氧缺血组(同时存在出生窒息及宫内窘迫)的原纤维酸性蛋白(GFAP)标记的星形胶质细胞的变化。结果生后24h内死亡组GFAP标记的星形胶质细胞的增生程度显著高于生后24~72h内死亡组、生后72h~6d死亡组,差异有统计学意义(P0.05);宫内窘迫组(慢性缺氧缺血)、出生窒息组(急性缺氧缺血)、混合性缺氧缺血组(同时存在出生窒息及宫内窘迫)的原纤维酸性蛋白(GFAP)标记的星形胶质细胞的变化比较,差异有统计学意义(P0.05)。结论缺氧缺血性脑病患儿的神经病理改变与HIE的病程、病情严重程度密切相关。     

脑缺血诱发星形胶质细胞粒细胞集落刺激因子受体和胶质细胞源性神经营养因子的表达

目的 探讨粒细胞集落刺激因子(G-CSF)在缺血脑保护中的作用机制.方法 制作大鼠大脑中动脉栓塞模型,7 d后断头取脑做冰冻切片,用免疫荧光双标的方法观察缺血周边区与假手术组相同部位G-CSF受体、胶质细胞源性神经营养因子(GDNF)和微管相关蛋白2(MAP2)、神经胶质原酸性蛋白(GFAP)的共表达情况.结果 G-CSF受体和GDNF在正常大鼠脑内广泛表达于神经元,不表达于星形胶质细胞;但在缺血周边区,星形胶质细胞亦部分表达G-CSF受体和GDNF.在正常脑组织,大部分G-CSF受体阳性的细胞也表达GDNF.结论 脑缺血可诱导缺血周边区星形胶质细胞表达G-CSF受体和GDNF,推测缺血后的内源性神经保护作用可能与缺血周边区星形胶质细胞的G-CSF受体表达以及GDNF产生有关.     

缺血后海马高迁移率族蛋白与星形胶质纤维酸蛋白表达及相关性

目的研究大鼠局灶性脑缺血再灌注星形胶质纤维酸蛋白(GFAP)与高迁移率族蛋白(HMGB1)在海马CA1区表达变化,探讨二者之间的关系。方法采用大脑中动脉栓塞2h制备SD大鼠脑缺血模型,60只雄性SD大鼠随机分为假手术组、缺血再灌注组,按1d、3d、7d、14d、28d时间点再分5个亚组,各时间点处死取脑,用免疫组化和荧光双标结合共聚焦扫描的方法来检测高迁移率族蛋白和星形胶质纤维酸蛋白在脑内海马CA1区表达变化。结果不同时间点缺血再灌注组GFAP、HMGB1表达均高于同时期的假手术组(P<0.05)。缺血再灌注组星形胶质细胞1d、3d、7d逐渐激活增生,7d达到高峰,14d开始下降;HMGB1在1d、3d、7d、14d是表达增加,14d达高峰,28d下降(与前一时间点比较P<0.05)。缺血再灌注组GFAP和HMGB1表达具有相关性(P<0.05),存在HMGB1和GFAP共定位细胞。结论脑缺血再灌注后,海马CA1区HMGB1增加与星形胶质细胞激活成正相关,过度表达的HMGB1和增殖的星形胶质细胞可能与缺血再灌注后神经元的迟发性损伤有关。     

槲皮素对氧化应激大鼠星形胶质细胞的保护作用

目的 研究槲皮素对氧化应激损伤后大鼠星形胶质细胞的保护作用. 方法 采用终浓度为2 mmol/L的H2O2作用于体外原代培养的大鼠胶质细胞6 h,以诱导氧化应激.实验分为正常对照组、H2O2组、槲皮素+H2O2组.不同浓度(0、50、100、200μmol/L)槲皮素预处理24h后,应用速率法和LIVE/DEAD检测试剂盒分别检测氧化应激胶质细胞的乳酸脱氢酶(LDH)释放率以及细胞存活率的变化. 结果 终浓度为2 mmol/L的H2O2作用6 h即可造成细胞损伤,LDH释放率由对照组的(3.89±1.89)%增至(90.27±2.68)%,较对照组明显增多,细胞存活率由对照组的(99.25±0.08)%降至(59.73%±9.92)%,较对照组明显降低,差异均有统计学意义(P<0.05).槲皮素预处理后细胞LDH释放率降低,50、100、200 μmol/L浓度的槲皮素组LDH释放率分别减少到(48.19±13.98)%、(27.81±9.33)%和(18.13±8.28)%,与H2O2组比较差异有统计学意义(P<0.05);槲皮素预处理同时能提高细胞存活率,50、100、200 μmol/L浓度的槲皮素组细胞存活率分别提高至(86.80±3.62)%、(88.32±5.77)%和(91.18±3.03)%,与H2O2组比较差异均有统计学意义(P<0.05). 结论 槲皮素预处理对氧化应激大鼠胶质细胞有一定的保护作用.     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

Hypoxia-induced astrocytic reaction and increased vascular permeability in the rat cerebellum

Hypoxia is an important factor linked to induction of vascular leakage and formation of brain edema. In this connection, astrocytes associated closely with the blood vessels are deemed to be involved. This study investigated the response of astrocytes to hypoxia in the adult rat cerebellum, and along with this, the integrity of the blood-brain barrier (BBB) was assessed using fluorescent and electron dense tracers. In rats subjected to hypoxia, mRNA and protein expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and aquaporin-4 (AQ4) was significantly increased. VEGF and AQ4 immunoreactive cells were identified as astrocytes by double immunofluorescence labeling. Increased VEGF tissue concentration and astrocytic swelling as observed in hypoxic rats were reduced after melatonin administration. Following intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in hypoxic rats but not in the controls indicating that functional integrity of BBB is compromised in hypoxia/reoxygenation. Enhanced gene and protein expression of VEGF may contribute to increased permeability of blood vessels. AQ4, a water transporting protein, is upregulated in astrocytes in hypoxia suggesting the cells are involved in edema formation. To this end, melatonin may be beneficial in reducing edema as it reduced VEGF concentration and, hence, vascular permeability.     

Inhibition of leptin-induced IL-1beta expression by glucocorticoids in the brain

Leptin is an important circulating signal for the regulation of food intake and body weight. Glucocorticoids were suggested to play a physiological role in the feedback inhibition of immune/inflammatory responses. In the present study, we examined whether these neuroendocrine effects of glucocorticoids are linked to changes in the leptin-induced expression of IL-1beta and STAT3 activation in the brain. Intravenous injection of leptin induced IL-1beta expression in the hypothalamus. Pretreatment with dexamethasone dose dependently inhibited leptin-induced IL-1beta expression in the hypothalamus. Moreover, dexamethasone inhibited leptin-induced IL-1beta expression in the primary cultured glial cells. In contrast, pretreatment with dexamethasone did not inhibit leptin-induced STAT3 phosphorylation in the hypothalamus. These effects of dexamethasone may not be due to the change in the expression level of the leptin receptor Ob-Ra and Ob-Rb isoforms. Therefore, it is suggested that glucocorticoid negatively regulates leptin-induced IL-1beta expression in the brain.     

Glial vascular endothelial growth factor overexpression in rat brainstem under tolerable hypoxia: evidence for a central chemosensitivity

The ventilatory response to hypoxia is mediated by peripheral inputs arising from the arterial chemoreceptors. In their absence, hypoxic adaptation can be achieved, possibly as a result of central cellular reorganization. To study this reorganization, we used chemodenervated rats to investigate the expression and localization of vascular endothelial growth factor (VEGF) in the brainstem. VEGF is a target gene of hypoxia-inducible factor (HIF) that is responsible for the morphofunctional remodeling induced by hypoxia. Intact and chemodenervated rats were subjected to normoxia or hypoxia for 6 hr (10% O(2) in N(2)). VEGF protein was quantified in micropunches of brainstem tissue. Only chemodenervated animals showed an increased VEGF expression in response to hypoxia, whereas, in normoxia, VEGF expression was not modified by chemodenervation. The same hypoxic condition was repeated for 8 days before immunocytochemical staining with anti-VEGF; antiglial fibrillary acidic protein (GFAP), a marker of astrocytes; and anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells. Confocal analysis showed a cellular colocalization of GFAP and VEGF, indicating that VEGF was overexpressed predominantly in astrocytes. Increased RECA-1 immunolabeling indicated an enhanced angiogenesis in chemodenervated rats subjected to hypoxia. These results indicate that glial cells and the vascular network contribute to the brainstem remodeling. The peripheral chemodenervation reveals a central O(2) chemosensitivity involving a cascade of gene expression triggered by hypoxia, which in intact animals may act synergically with peripheral chemosensory inputs.     

Cerebellar alterations induced by chronic hypoxia: an immunohistochemical study using a chick embryonic model

A model of fetal aerogenic hypoxia was developed in which fertilized chicken eggs were half-painted with melted wax and incubated under normal conditions. The cerebellum of the hypoxic chick embryos at a later stage of development (E18-20) was analyzed immunochemically. Hypoxic insult resulted in considerable neurocytological deficits of the Purkinje cells and altered glial fibrillary acid protein (GFAP) immunoreactivity in the fetal cerebellum. Purkinje cells in the hypoxic embryos were marked by small cell size, poorly developed dendrites, low cell density, deletion and ectopia. On the other hand, enhanced GFAP immunoreactivity was found in astrocytes and Bergmann glia of the hypoxic embryos. Our results indicate that chronic hypoxia in the chick fetus can cause severe disorders of neuronal development as well as glial activation. We suggest that our hypoxic model of chick embryos could be an accessible animal model for further elucidating fetal hypoxia.     

大鼠脑梗死后乏氧组织长时间存在与星形胶质细胞的关系

目的 探讨大鼠脑梗死后乏氧组织的长时间存在与星形胶质细胞的关系.方法 利用大鼠脑缺血1.5 h再灌注(1.5 h IR组)和持续缺血(PI组)的大脑中动脉闭塞模型,采用EF5和胶质纤维酸性蛋白(GFAP)免疫荧光双标方法观察乏氧组织和星形胶质细胞增殖活化情况,比较两组术后1、3、7、14d缺血侧大脑半球皮层的星形胶质细胞GFAP荧光强度以及与乏氧组织的关系.结果 1.5 h IR组术后1、3、7、14 d均见乏氧组织存在,PI组乏氧组织仅存在3 d.各观察时间点乏氧组织内的GFAP荧光强度均较周围区域高,差异有统计学意义(P<0.05).随着时间的延长,两组的GFAP荧光强度均不断增强,在术后7 d达到高峰,14 d时下降,差异有统计学意义(P<0.05);且各时间点的1.5 h IR组的GFAP荧光强度均高于PI组,差异有统计学意义(P<0.05).结论 脑梗死后星形胶质细胞在乏氧组织内增殖活化尤为明显,乏氧组织的长时间存在与星形胶质细胞密切相关.     

瘦素对局灶性脑缺血后内质网应激相关蛋白的作用

目的探讨瘦素对大鼠局灶性脑缺血后内质网应激相关蛋白的影响。方法 SD大鼠40只,随机分为假手术组、脑缺血组、脑缺血瘦素预处理组,线栓法制备大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型,在闭塞血管前3 h皮下注射瘦素,于闭塞血管6 h后采用Longa 5分制量表进行神经功能评分、称量体重及脑水肿变化,并灌注取脑,采用免疫荧光化学法检测内质网应激相关蛋白葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)及C/EBP同源蛋白(C/EBP-homologous protein,CHOP)表达。结果脑缺血瘦素预处理组和脑缺血组体重变化未见差异(P0.05);脑缺血瘦素预处理组神经功能评分(1.90±0.31 vs.2.50±0.52,P0.05)和脑水肿程度(3.60±0.52 vs.7.70±0.94,P0.001)明显轻于脑缺血组,同时脑缺血瘦素预处理组GRP78表达明显高于脑缺血组(48.69±5.06 vs.35.78±4.35,P0.01),CHOP表达明显低于脑缺血组(38.81±5.34 vs.60.24±4.11,P0.01)。结论瘦素可减少神经功能缺损并可能与其上调GRP78蛋白,下调CHOP蛋白来减弱脑缺血导致的内质网应激有关。     

缺氧复氧大鼠海马星形胶质细胞凋亡及Bcl-2和Bax蛋白表达的时间进程变化及异丙酚干预效应

BACKGROUND： Cerebral hippocampal astrocytes are more sensitive.to ischemic injury than neurons. Hypoxic-ischemic brain injury induces profound astrocyte apoptosis, and propofol may protect against astrocyte apoptosis.
OBJECTIVE： To verify the protective effects of propofol against astrocyte apoptosis and to investigate anti-apoptotic Bcl-2 and pro-apoptotic Bax expression in primary cultures of rat hippocampal astrocytes exposed to hypoxia-reoxygenation for different periods of time following propofol treatment.
DESIGN, TIME, AND SETTING： In vitro neural immunocytochemistry was performed at the Central Laboratory of Yunyang Medical College between September 2007 and March 2008.
MATERIALS： A total of 30 Wistar rats, aged 1-3 days, wJth equal numbers of males and females, were included for isolation and culture of .hippocampal astrocytes.
METHODS： Hippocampal astrocytes were purified and cultured for 3 weeks and treated with four culture conditions： 50 μL Hank＇s solution （normal control）; 0.2 mL/L Intralipid; 50 μL Hank＇s solution for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 or 72 hours; propofol （250 μmol/L final） for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 and 72 hours.
MAIN OUTCOME MEASURES： （1） Morphologic changes in hippocampal astrocytes. （2） Levels of astrocyte apoptosis and Bcl-2 and Bax expression.
RESULTS： Hypoxia and reoxygenation increased apoptosis over time, with Bcl-2 expression peaking at 24 hours and decreasing gradually （P 〈 0.01 ）; Bax expression peaked at 72 hours （P 〈 0.01）; the ratio of Bcl-2/Bax was 1.4, 0.8, and 0.6, respectively, at 24, 48 and 72 hours. Non-apoptotic astrocytes showed significant proliferation and swelling. Propofol treatment decreased apoptosis after hypoxia-reoxygenation （P 〈 0.01）, as well as Bct-2 and Bax expression （P 〈 0.05, P 〈 0.01）, with Bcl-2/Bax ratios of 1.6-1.8. Propofol treatmentalso blocked astrocyte proliferation and swelling. No apoptotic cells or Bcl-2/Bax expression was detected in astrocytes cultured in Hank＇s or Intralipid solution.
CONCLUSION： Propofol protects astrocytes against injury caused by hypoxia and reoxygenation via a mechanism that involves maintaining high ratios of Bcl-2/Bax.