亚低温对脊髓损伤后反应性星形胶质细胞增生的影响

目的：观察在不同温度条件下脊髓星形胶质细胞划痕损伤活化后的形态和活性改变,以探讨亚低温对脊髓损伤后反应性星形胶质细胞增生的影响。方法：体外原代培养新生SD大鼠脊髓星形胶质细胞,以划痕实验制备反应性星形胶质细胞。亚低温选择33℃,细胞培养48 h。实验分为对照组、划痕组、亚低温组和划痕+亚低温组。各组在相应的时间点观察细胞形态,采用免疫荧光染色方法检测Nestin阳性率,MTT比色法观察细胞活性,PI染色方法观察细胞凋亡程度。结果：与对照组和亚低温组相比,划痕组和划痕+亚低温组细胞胞体肥大,周围突起增多、延展以及胞浆丰富,细胞生长率明显升高。与划痕组相比,划痕+亚低温组细胞变化减慢,周围突起减少,细胞长入划痕处所需时间增加,细胞Nestin阳性率、PI阳性率和细胞生长率明显降低,各结果差异显著（P<0.01）。结论：划痕损伤后星形胶质细胞活化为反应性星形胶质细胞并会增生,亚低温明显抑制脊髓反应性星形胶质细胞的活化增生,并可以抑制星形胶质细胞的凋亡。     

L-Glu促进纯化培养的海马和皮质星形胶质细胞增殖的研究

目的研究L-Glu对纯化培养的皮质和海马星形胶质细胞促增殖作用的机制.方法将纯化培养的星形胶质细胞分为7组:①无血清培养基组,②含L-Glu(1mmol/L)的无血清培养基组,③含MCCG(100μmol/L)的无血清培养基组,④含MPEP(100μmol/L)的无血清培养基组,⑤含L-Glu MCCG(分别为1mmol/L,100μmol/L)的无血清培养基组,⑥含L-Glu MPEP(分别为1mmol/L,100μmol/L)的无血清培养基组,⑦含L-Glu MCCG MPEP(分别为1mmol/L,100μmol/L及100μmol/L)的无血清培养基组.流式细胞仪检测细胞周期的变化.结果 L-Glu促进纯化培养的星形胶质细胞的增殖,单独加入mGluRs的竞争性拮抗剂MCCG或MPEP则L-Glu的促增殖作用减弱,同时加入两种拮抗剂则L-Glu的促增殖作用消失.结论 L-Glu通过作用于mGluR3和mGluR5促进星形胶质细胞的增殖,两者可能在星形胶质细胞增殖方面具有协同作用.     

治疗脊髓损伤之建立星形胶质细胞模型的研究进展

脊髓损伤(spinal cord injury,SCI)是一种极为复杂的破坏性疾病,一旦脊髓损伤发生,治疗棘手,对患者家庭、国家带来巨大的经济、社会负担。近年来,通过建立大鼠脊髓损伤细胞相关模型,对于脊髓损伤的病因病机治疗等方面有了进一步的认识,而星形胶质细胞模型的建立对脊髓损伤治疗有深远意义。研究发现,星形胶质细胞作为靶细胞通过血-脑脊液屏障直接或间接对脊髓损伤有双向调控作用。本文通过对近年来星形胶质细胞模型培养制备方案等研究进行总结,以期为建立一个客观化、定量化、可模拟化的星形胶质细胞模型提供指导对脊髓损伤的治疗提供新的思路。     

新生大鼠脊髓星形胶质细胞体外培养方法

星形胶质细胞是中枢神经系统主要的胶质细胞 ,对神经元具有绝缘、营养、保护和支持作用。它们在中枢神经系统损伤和修复中也具有重要的作用 ,一方面星形胶质细胞可合成神经营养因子 ,促进神经再生[1～ 3] ,另一方面合成神经生长抑制因子 ,如硫酸软骨素蛋白多糖等 [4 ] ,抑制神经再生 ,尤其是损伤恢复后期形成星胶瘢痕被认为是神经再生的机械性障碍。脊髓损伤后的修复一直是神经科学领域研究的一个重要课题 ,随着分子生物学和精密方法、仪器的发展 ,离体研究被越来越多地采用。星形胶质细胞是神经再生微环境中的主要成分 ,深入研究星形胶质细…     

电针对脊髓损伤星形胶质细胞增生及其NGF表达的影响

目的研究脊髓损伤后电针治疗对星形胶质细胞增生及其内源性神经生长因子(nerve growth factor,NGF)表达的影响.方法选用成年雌性Wistar大鼠,随机分为3组.A组为正常对照组,B组、C组为下胸段脊髓不完全损伤.B组损伤后不治疗,C组损伤后给予督脉电针治疗.损伤后3 d、1 、2或4周应用免疫组化染色分别观察损伤脊髓胶质原纤维酸性蛋白(glial fibroblast acid protein,GFAP)和NGF表达的变化.结果 B组术后3 d,GFAP阳性细胞明显增多, 2周后开始减少,4周时仍有较多的阳性细胞;C组GFAP阳性细胞明显少于B组,1周时达高峰.脊髓损伤后NGF表达呈逐渐增加的趋势.C组NGF的表达明显高于B组,且一直保持在较高水平.NGF阳性细胞大部分与GFAP阳性细胞形态相似.结论电针治疗能减少星形胶质细胞增生,促进内源性NGF的合成,从而创造了有利于神经再生的微环境.     

星形胶质细胞

目录一、星形胶质细胞的生物学特性(一 )星形胶质细胞的异质性(二 )胶质网络二、星形胶质细胞的功能(一 )分泌功能(二 )星形胶质细胞与神经的发育及再生(三 )星形胶质细胞具有对神经元微环境调控的能力(四 )免疫功能与血脑屏障调控三、星形胶质细胞功能的新近进展(一 )星形胶质细胞也具有可兴奋性(二 )星形胶质细胞与神经元的通讯或对话(三 )在突触形成和突触可塑性中的作用(四 )星形胶质细胞与神经发生胶质细胞是神经系统内数量众多的一大类细胞群体 ,约占中枢神经系统 (CNS)细胞总数的 90 % ,星形胶质细胞 (astrocyte)是其中主要的组成…     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化

已知miR-144与细胞活化和增殖有关,然而其具体分子机制尚不明确。本研究发现miR-144通过靶向GRK5促进脊髓星形胶质细胞的活化。运用real-time PCR检测脊髓损伤和正常大鼠的脊髓组织及其脊髓星形胶质细胞中miR-144的表达,发现与正常的组织和细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低;Western印迹检测到脊髓损伤大鼠的星形胶质细胞中GFAP蛋白的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性试剂盒分析发现miR-144显著提高了SOD和GSH活性;生物学信息分析和萤光素酶报告基因检测结果显示miR-144能靶向结合GRK5,并下调GRK5的表达;MiR-144 mimic转染或miR-144 mimic与pcDNA-GRK5共转染脊髓损伤的星形胶质细胞,发现miR-144转染能通过激活NF-κB通路消除pcDNA-GRK5引起的细胞活化抑制。综上所述,miR-144通过靶定结合癌基因GRK5来促进脊髓星形胶质细胞细胞的活化。     

Olomoucine对星形胶质细胞增殖及活化分泌的影响

目的观察细胞周期抑制剂olomoucine对培养星形胶质细胞机械损伤后增殖和活化分泌的影响。方法建立体外培养大鼠纯化的AS物理损伤模型（划痕损伤模型）,分为对照组、划痕组和olomoucine干预组。利用免疫荧光细胞化学方法观察损伤后GFAP表达;利用RT-PCR观察细胞因子IL-6和TNF-α的表达情况。结果划痕损伤刺激能使培养AS反应性增生活化。损伤早期出现IL-6、TNF-α表达水平增高,从损伤后12h开始出现损伤边缘区AS胞体肥大,数目增加;给予olomoucine干预后,细胞数目减少、体积明显减小,损伤后IL-6和TNF-α表达也显著下降。结论损伤刺激可促使AS活化,并发生反应性胶质增生;CDK选择性细胞周期抑制剂olomoucine可以通过调控细胞周期,有效抑制损伤后星形胶质细胞的反应性增生,并能对AS的活化起到抑制作用。     

疼痛研究的新亮点:星形胶质细胞

一直以来疼痛被认为仅仅是由神经元调节的。目前的研究表明,星形胶质细胞与疼痛有密切的关系。星形胶质细胞通过许多重要功能如参与信号转导、被激活而表现出激活的特性,如释放促炎性因子、神经营养因子等,在疼痛调节过程中发挥重要作用。对星形胶质细胞与疼痛关系的研究,必将为疼痛机制的阐明及疼痛治疗提供新的思路。     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化北大核心CSCD

尽管miR-144与细胞活化、增殖有关,但其对脊髓星形胶质细胞的作用尚不明确。本文旨在探究正常和脊髓损伤(spinal cord injury,SCI)组织和细胞中miR-144表达,以及miR-144能否通过靶向调节G蛋白偶联受体激酶5(GRK5)上调脊髓星形胶质细胞活化。实时定量PCR(RT-q PCR)和Western印迹结果揭示,与正常的组织/细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低,而GRK5的表达升高;脊髓损伤大鼠的星形胶质细胞中胶质原纤维酸性蛋白(GFAP)的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示,转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性分析发现,miR-144显著提高SOD和GSH活性;萤光素酶报告基因检测结果证明,miR-144能靶向结合GRK5,下调GRK5表达。miR-144 mimic转染或miR-144 mimic与pc DNA-GRK5共转染脊髓损伤的星形胶质细胞后,我们发现,miR-144转染可通过激活NF-κB通路消除GRK5对细胞活化的抑制作用。综上所述,miR-144通过靶向抑制GRK5促进脊髓星形胶质细胞的活化。     

Immunocytochemical characterization of a new marker of fibrous and reactive astrocytes

A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin. Received: 2 May 1995 / Accepted: 31 July 1995     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

High‐mobility group box 1 from reactive astrocytes enhances the accumulation of endothelial progenitor cells in damaged white matter

High‐mobility group box 1 (HMGB1) was initially described as a damage‐associated‐molecular‐pattern (DAMP) mediator that worsens acute brain injury after stroke. But, recent findings suggest that HMGB1 can play a surprisingly beneficial role during stroke recovery by promoting endothelial progenitor cell (EPC) function and vascular remodeling in cortical gray matter. Here, we ask whether HMGB1 may also influence EPC responses in white matter injury. The standard lysophosphatidylcholine (LPC) injection model was used to induce focal demyelination in the corpus callosum of mice. Immunostaining showed that within the focal white matter lesions, HMGB1 was up‐regulated in GFAP‐positive reactive astrocytes, along with the accumulation of Flk1/CD34‐double‐positive EPCs that expressed pro‐recovery mediators such as brain‐derived neurotrophic factor and basic fibroblast growth factor. Astrocyte–EPC signaling required the HMGB1 receptor RAGE as treatment with RAGE‐neutralizing antibody significantly decreased EPC accumulation. Moreover, suppression of HMGB1 with siRNA in vivo significantly decreased EPC numbers in damaged white matter as well as proliferated endothelial cell numbers. Finally, in vitro cell culture systems confirmed that HMGB1 directly affected EPC function such as migration and tube formation. Taken together, our findings suggest that HMGB1 from reactive astrocytes may attract EPCs to promote recovery after white matter injury.     

Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

Protective effect of leptin-mediated caveolin-1 expression on neurons after spinal cord injury

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca²⁺, and this Ca²⁺ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca²⁺ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca²⁺ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

SHANK1 is differentially expressed during development in CA1 hippocampal neurons and astrocytes

Recent studies have strongly suggested a role for the synaptic scaffolding protein SHANK1 in normal synaptic structure and signaling. Global SHANK1 knockout (SHANK1?/?) mice demonstrate reduced dendritic spine density, an immature dendritic spine phenotype and impairments in various cognitive tasks. SHANK1 overexpression is associated with increased dendritic spine size and impairments in fear conditioning. These studies suggest proper regulation of SHANK1 is crucial for appropriate synaptic structure and cognition. However, little is known regarding SHANK1's developmental expression in brain regions critical for learning. The current study quantified cell specific developmental expression of SHANK1 in the hippocampus, a brain region critically involved in various learning paradigms shown to be disrupted by SHANK1 dysregulation. Consistent with prior studies, SHANK1 was found to be strongly co‐expressed with dendritic markers, with significant increased co‐expression at postnatal day (P) 15, an age associated with increased synaptogenesis in the hippocampus. Interestingly, SHANK1 was also found to be expressed in astrocytes and microglia. To our knowledge, this is the first demonstration of glial SHANK1 localization; therefore, these findings were further examined via a glial purified primary cell culture fraction using magnetic cell sorting. This additional analysis further demonstrated that SHANK1 was expressed in glial cells, supporting our immunofluorescence co‐expression findings. Developmentally, astroglial SHANK1 co‐expression was found to be significantly elevated at P5 with a reduction into adulthood, while SHANK1 microglial co‐expression did not significantly change across development. These data collectively implicate a more global role for SHANK1 in mediating normal cellular signaling in the brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 363–373, 2018