LPS诱导海马星形胶质细胞活化与活化的星形胶质细胞对海马神经元的影响

探讨脂多糖(Lipopolysaccharide,LPS)对长时间存活大鼠海马内星形胶质细胞的反应以及对神经元的影响。方法:本实验用10只健康成年雄性SD大鼠,海马CA3区注射LPS 10μ1．7和14d后,尼氏染色观察神经元的变化,免疫组织化学染色结合图像分析方法观察海马CA3区注射部位胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)、的表达变化。结果:脂多糖可促进海马星形胶质细胞的活化,但并不能引起海马区神经元的损伤。结论:星形胶质细胞在脑损伤后的脑内炎症反应起了一定的作用,但并不能引起神经元的损伤。     

模拟高原低氧对大鼠脑内神经胶质细胞的影响

目的：观察模拟高原低氧对成年大鼠脑内胶质细胞的影响。方法：大鼠在模拟海拔4000m高原低压舱内连续暴露1、3、7d,胶质原纤维酸性蛋白（GFAP）与Griffoniasimplicifolia同功凝集素（GSA-IB4）组织细胞化学分别显示星形胶质细胞与小胶质细胞。结果：GFAP与GSA-IB4阳性细胞在低氧后3．7d两个时相显著增多,主要分布于新皮层、海马、纹状体及室管膜下层等区域。结论：模拟高原低氧能引起大鼠脑内星形胶质细胞与小胶质细胞的显著活化。     

低氧预适应对离体星形胶质细胞低氧耐受性的影响

目的：研究低氧预适应对体外培养的星形胶质细胞低氧耐受性的影响。方法：体外培养的鼠脑星形胶质细胞,随机分为对照组（control,C组）,低氧损伤组（hypoxia,H组）,低氧预适应组（hypoxic preconditioning,HP组）,通过检测细胞MTT代谢变化、凋亡发生和形态学观察探讨低氧预适应对星型胶质细胞低氧损伤的保护作用;免疫细胞化学方法分析Bcl-2和Bax的表达差异。结果：与低氧组相比,HP48、HP72组MTT代谢活性较高。免疫细胞化学结果提示低氧预适应组Bcl-2表达高于低氧损伤组,低氧预适应组Bax表达低于低氧损伤组。结论：低氧预适应对大鼠星形胶质细胞低氧损伤有保护作用,可能与Bax表达受抑,维持Bcl-2表达有关,通过对抗凋亡程序的发展产生保护作用。     

小胶质细胞和星形胶质细胞相互作用在中枢神经系统疾病中的作用机制研究进展

小胶质细胞和星形胶质细胞是中枢神经系统主要的两种胶质细胞,两者各自在中枢神经系统中扮演着重要的角色。本文主要从小胶质细胞和星形胶质细胞相互交流这一新的角度,概述两种胶质细胞相互作用方式及在中枢神经系统中的研究现状,并进一步阐明两者相互作用在各种中枢神经系统疾病的功能及机制,旨在为进一步了解这两种胶质细胞在中枢神经系统疾病的作用机理提供理论依据、为治疗相关中枢系统疾病提供新思路。     

低氧促进脂多糖诱导小胶质细胞CXCL10表达

本文旨在观察低氧处理对脂多糖(lipopolysaccharide, LPS)诱导的小胶质细胞CXC趋化因子配体10 (CXC-chemokine ligand-10,CXCL10)表达的影响,并探讨其作用机制。将C57BL/6J小鼠随机分为对照组、低氧组、LPS组以及低氧联合LPS组,LPS组腹腔注射0.5 mg/kg LPS,低氧组放置于低压低氧舱(模拟海拔6 000 m)中,处理6 h后收集血清和海马组织样品。用ELISA法检测血清和海马组织中CXCL10的含量。用低氧(1%O2)和/或LPS (100 ng/mL)刺激小胶质细胞系BV2和原代小胶质细胞6 h,用实时定量PCR检测细胞CXCL10 mRNA表达水平,用ELISA检测细胞培养上清中CXCL10的含量,用Western blot检测核因子κB (nuclear factorκB, NF-κB)信号通路相关蛋白p65和IκBα的表达。另外,用小分子化合物PDTC阻断NF-κB信号通路后检测BV2细胞CXCL10 mRNA表达水平。结果显示,在LPS诱导的小鼠炎症模型中,低氧处理可促进LPS对小鼠血清和海马组织中CXCL...     

缺血缺氧对体外培养星形胶质细胞细胞活化和细胞周期的影响

目的观察缺血缺氧损伤对星形胶质细胞细胞活化和细胞周期的影响。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胶质细胞纤维酸性蛋白(GFAP)及细胞周期蛋白cyclinD1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,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。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,而cyclinD1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;cyclinD1参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。     

缺氧对大鼠大脑皮层星形胶质细胞Inos Mrna表达的影响

目的：观察缺氧和谷氨酸对星形胶质细胞诱导型一氧化氮合酶（iNOS)mRNA表达的影响,探讨大脑星形胶质细胞在缺氧性脑血管扩张反应中的作用。方法：取新生Wistar大鼠大脑皮层进行星形胶质细胞原代、传代培养,分为四组：（1）对照组;（2）谷氨酸组;（3）缺氧组;（4）缺氧＋谷氨酸组。每一组包括5个时相点：0h、3h、6h、12h、24h(以缺氧后开始记时）。于（2）和（4）组加入100μmol/Lr L-谷氨酸。（3）和（4）组用95％N2／5％CO2的混合气体缺氧。提取总RNA,用RT－PCR技术检测iNOS mRNA的表达量。结果：对照组和谷氨酸组各时相点未见星形胶质细胞iNOS mRNA表达。缺氧组与缺氧＋谷氨酸组iNOSmRNA于6h开始显著增高,以后更为显著（24h内）。缺氧＋谷氨酸组iNOSmRNA表达的幅度显著高于缺氧组。结论：缺氧及缺氧＋谷氨酸可使iNOSmRNA表达增强,后者催化合成一氧化氮,作用于脑血管平滑肌,可能是缺氧性脑血管扩张的重要机制之一。     

原代培养星形胶质细胞和小胶质细胞的特性与鉴定

本研究旨在明确原代培养的星形胶质细胞和小胶质细胞不同代次的生长特性,优化高效获取状态一致细胞的技术方法。将新生乳鼠的脑组织进行原代分离培养胶质细胞,通过细胞增殖检测试剂盒（cell counting kit-8,CCK-8）测定混合胶质细胞增殖曲线,使用流式细胞术检测两类细胞比例,并通过免疫荧光染色鉴定两类胶质细胞分型情况。生长曲线显示P0和P1代混合胶质细胞增殖活力最好;通过170 r/min机械振摇30 min可获得97.3%的高纯度小胶质细胞,该纯化方法得到的P0、P1、P2代离子钙接头蛋白-1（ionized calcium-binding adapter molecule 1,Iba-1）阳性小胶质细胞的形态及其M1、M2表型比例无代次差别;通过星形胶质细胞表面抗原-2（astrocyte cell surface antigen-2,ACSA-2）磁珠抗体分选的方法可获得纯度达到95.7%的星形胶质细胞,该纯化方法得到的P0、P1、P2代胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性星形胶质细胞的形态及其A1、A2表型比例无代次差别。本研究详述了原代分离培养的小胶质细胞和星形胶质细胞的生长特点,证明了获取两类胶质细胞的最佳代次,优化了获取两类胶质细胞的技术方法,验证了连续培养两代不会影响其功能表型。本结果为研究神经系统炎症相关疾病的分子机制提供了技术支撑。     

星形胶质细胞的生物学功能

人类大脑由两类细胞组成:一类是神经元,另一类是神经胶质细胞。神经胶质细胞的数量约为神经元的10倍,但其作用长期以来一直被认为仅限于在神经元之间充当填充物,填满大脑中的剩余空间,同时为神经元提供营养。但近年来认识到神经胶质细胞的主要成员星形胶质细胞能够感知外界刺激,它的反应选择性甚至高于相邻神经元。神经元的反应活动很多都要经过星形胶质细胞的介导才能完成。本文介绍了星形胶质细胞在神经调制、突触调节和神经血管系统偶联方面的一些新进展,以期在不久的将来对星形胶质细胞的功能有更深入的了解,并能应用于临床实践。     

重组反义GFAP逆转录病毒对星形胶质细胞在胶质瘢痕增生中作用的研究

本文研究了脑穿刺损伤后伤灶组织中大胶质细胞的变化、性激素对脑损伤后星形胶质细胞反应的影响,以及反义胶质原纤维酸性蛋白（ＧＦＡＰ）逆转录病毒表达载体对Ａｓｔ形态结构,反应性胶质化及胶质瘢痕形成的作用。结果表明,胶质瘢痕中增生的大胶质细胞主要是Ａｓｔ,ＧＦＡＰ对维持Ａｓｔ的形态结构及功能具有重要作用;少突胶质细胞在胶质瘢痕形成过程中不是反应活跃的细胞成分;性激素对Ａｓｔ的反应性胶质化有一定程度的抑制作     

BNIP3 mediates pre‐myelinating oligodendrocyte cell death in hypoxia and ischemia

Developing oligodendrocytes, collectively termed ‘pre‐myelinating oligodendrocytes’ (preOLs), are vulnerable to hypoxic or ischemic insults. The underlying mechanism of this vulnerability remains unclear. Previously, we showed that Bcl‐2?E1B‐19K‐interacting protein 3 (BNIP3), a proapoptotic member of the Bcl‐2 family proteins, induced neuronal death in a caspase‐independent manner in stroke. In this study, we investigated the role of BNIP3 in preOL cell death induced by hypoxia or ischemia. In primary oligodendrocyte progenitor cell (OPC) cultures exposed to oxygen–glucose deprivation, we found that BNIP3 was upregulated and levels of BNIP3 expression correlated with the death of OPCs. Up‐regulation of BNIP3 was observed in preOLs in the white matter in a neonatal rat model of stroke. Knockout of BNIP3 significantly reduced death of preOLs in the middle cerebral artery occlusion model in mice. Our results demonstrate a role of BNIP3 in mediating preOLs cell death induced by hypoxia or ischemia, and suggest that BNIP3 may be a new target for protecting oligodendrocytes from death after stroke.

     

p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stroke.     

Nitric oxide induces BNIP3 expression that causes cell death in macrophages

Nitric oxide (NO) is involved in many physiological processes and also causes pathological effects by inducing apoptosis. It can enhance or suppress apoptosis depending on its concentration and the cell type involved. In this report, we used cDNA microarray analysis to show that SNAP, an NO donor, strongly induces Bcl-2/adenovirus E1B 19kDa-interacting protein 3 (BNIP3) in macrophages. BNIP3 is a mitochondrial pro-apoptotic protein that contains a Bcl-2 homology 3 domain and a COOH-terminal transmembrane (TM) domain. Macrophages activated by LPS/IFN-gamma produce nitric oxide synthase 2 (NOS2) and release endogenous NO. Expression of BNIP3 was also induced in macrophages by LPS/IFN-gamma, and the induction was blocked by a NOS2 inhibitor, S-methyl-isothiourea. Peritoneal macrophages from NOS2-null mice failed to produce BNIP3 in response to LPS/IFN-gamma. We conclude that BNIP3 expression in macrophages is controlled by the intracellular level of nitric oxide. Overexpression of BNIP3 but not of BNIP3 deltaTM, a BNIP3 mutant without the TM domain and C-terminal tail, led to apoptosis of the cells. Promoter analysis showed that the region between -281 and -1 of the 5'-upstream enhancer region of murine BNIP3 was sufficient for NO-dependent expression of BNIP3.     

Tumor suppressor functions of BNIP3 and mitophagy

There is a growing realization that tumor cells rely on healthy mitochondria to promote their growth under changing microenvironmental stresses and do so by dynamically modulating both their mitochondrial mass and state of mitochondrial fusion. Our recent work adds to this appreciation by showing that the mitophagy receptor BNIP3 functions as a tumor suppressor in mammary tumorigenesis and also as a prognostic indicator of progression to metastasis in certain sub-types of human breast cancer.     

乳酸对培养大鼠大脑皮层神经元缺氧/复氧损伤的影响

目的:观察不同浓度乳酸对原代培养大鼠大脑皮层神经元缺氧/复氧(H/R)损伤的影响及其机制。方法:原代培养大鼠大脑皮层神经元缺氧8、12、24h后在培养液中添加不同浓度乳酸(终浓度分别为0、0.5、1.0、2.0、5.0mmol/L),复氧24h,以细胞存活率和乳酸脱氢酶(LDH)释放量为指标,观察不同浓度乳酸对神经元H/R损伤的影响,并以盐酸模拟乳酸解离的H 的作用,探讨乳酸对大脑皮层神经元H/R损伤影响的机制。结果:5.0mmol/L乳酸和盐酸引起常氧神经元损伤并加重神经元H/R损伤;1.0mmol/L乳酸对缺氧12h和24h神经元H/R损伤具有保护作用,相同浓度盐酸对神经元H/R损伤无影响。结论:较低浓度乳酸对神经元H/R损伤具有保护作用,其机制与H 的作用无关;较高浓度乳酸加重神经元H/R损伤,其机制可能包括H 的作用。     

Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3

An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

The antipsychotic spiperone attenuates inflammatory response in cultured microglia via the reduction of proinflammatory cytokine expression and nitric oxide production

Glial activation and neuroinflammatory processes play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and HIV dementia. Activated glia cells can secrete various proinflammatory cytokines and neurotoxic mediators, which may influence neuronal cell survival. Recent studies have demonstrated that glia cell-mediated neuroinflammation is also related to the pathophysiology of schizophrenia. In the present study, anti-inflammatory and neuroprotective effects of antipsychotics were investigated using cultured brain cells as a model. The results showed that spiperone significantly decreased the production of nitric oxide in lipopolysaccharide-stimulated BV-2 microglia cells, primary microglia and primary astrocyte cultures. Spiperone also significantly inhibited nitric oxide production in adenosine 5'-triphosphate (ATP)-stimulated primary microglia cultures. Spiperone markedly decreased the production of tumor necrosis factor-alpha in BV-2 microglia cells. Spiperone attenuated the expression of inducible nitric oxide synthase and proinflammatory cytokines such as interleukin-1beta and tumor necrosis factor-alpha at mRNA levels in BV-2 microglia cells. Spiperone inhibited nuclear translocation and DNA binding of the p65 subunit of nuclear factor kappa B (NF-kappaB), inhibitor of kappa B (IkappaB) degradation, and phosphorylation of p38 mitogen-activated protein kinase in the lipopolysaccharide-stimulated BV-2 microglia cells. Moreover, spiperone was neuroprotective, as the drug reduced microglia-mediated neuroblastoma cell death in the microglia/neuron co-culture. These results imply that the antipsychotic spiperone has anti-inflammatory and neuroprotective effects in the central nervous system by modulating glial activation.