腺苷类物质及其受体在反应性星形胶质化过程中的作用

反应性星形胶质化是各类脑损伤后的修复过程,但过度的胶质化会成为中枢神经突起再生的障碍。腺苷及其类似物作为神经递质对反应性星形胶质化有明显的影响,ATP及腺苷可通过各自的受体来促进或抑制星形胶质化腺苷受体P2X7,P2Y在星形胶质化的调节中起重要作用。对调节胶质化的信号通路的研究可为临床合理控制胶质化反应、促进神经功能的恢复提供新的思路。

体外培养大鼠星形胶质细胞受损后的激活与反应性胶质化

用鼠脑星形胶质细胞（ＡＳ）原代培养技术建立体外ＡＳ机械性损伤模型。以ｃ—Ｆｏｘｓ、ｂＦＧＦ、ＰＣＮＡ、ＧＦＡＰ—ｍＲＮＡ、ＧＦＡＰ和Ｍｙｏｓｉｎ作为观察指标研究反应性星形胶质化的形成机制。结果显示：１．ｃ—Ｆｏｓ蛋白于损伤后４５ｍｉｎ即有阳性表达，伤后２ｈ消失；２．损伤后２ｈ，损伤边缘的ＡＳ开始表达ｂＦＧＦ，１２ｈ达高峰，２ｄ后表达强度开始回落；３．损伤边缘的部分ＡＳ于损伤后２ｈ开始表达ＰＣＮＡ，伤后１ｄ，ＰＣＮＡ阳性的ＡＳ沿损伤边缘呈列兵式整齐排列，２ｄ后ＰＣＮＡ阳性的ＡＳ分布于损伤周围区域；４．损伤后４ｈ，损伤边缘的ＡＳ开始表达Ｍｙｏｓｉｎ，并逐渐增加，而且朝向损伤区胞浆突起的阳性表达强于背向损伤区的突起；５．损伤边缘的ＡＳ于损伤后６ｈ开始表达ＧＦＡＰ—ｍＲＮＡ，１ｄ达高峰，２ｄ开始回落，３ｄ则只在少数ＡＳ中可检出ＧＦＡＰ—ｍＲＮＡ；６．损伤后１ｄ，ＧＦＡＰ表达明显增强，胞体肥大并向损伤区伸出粗大突起，２ｄＧＦＡＰ达高峰，３ｄ肥大ＡＳ的胞体和突起覆盖损伤区；７．在体外ＡＳ机械性损伤模型上，在没有神经元和其它复杂因素影响的条件下，ＡＳ对损伤的主要反应是胞体的肥大、突起的粗大，并能独立形成反应性星形胶质化。

体外鼠脑星形胶质细胞损伤后c-fos蛋白、GFAP及其mRNA的动态表达

研究ｃ－ｆｏｓ蛋白、ＧＦＡＰ—ｍＲＮＡ和ＧＦＡＰ在鼠脑星形胶质细胞（ａｓｔｒｏｃｙｅ，ＡＳ）损伤后反应性星形胶质化时的动态变化。用ＡＳ原代培养技术建立体外ＡＳ机械损伤模型．并结合应用免疫组织化学和原位杂交方法．结果：（１） ｃ－ｆｏｓ蛋白于损伤后 ４５ ｍｉｎ即有阳性表达，伤后 ２ ｈ消失； （２损伤边缘的 ＡＳ于损伤后 ６ ｈ开始表达 ＧＦＡＰ－ｍＲＮＡ， １ｄ达高峰，３ ｄ则在损伤边缘少数 ＡＳ中可检出 ＧＦＡＰ－ｍＲＮＡ；（３）损伤后 １ｄ，ＧＦＡＰ表达明显增强，胞体肥大，粗大突起伸向损伤区形成反应性星形胶质化。结论：（ｌ） ＡＳ受损后原癌基因 ｃ－ｆｏｓ首先被激活，ｃ－ｆｏｓ蛋白呈－过性的表达，参与调节ＡＳ的激活；（２）ＡＳ损伤后，ＧＦＡＰ－ｍＲＮＡ的转录增加，ＧＦＡＰ表达增强是由于在转录水平上ＧＦＡＰ－ｍＲＮＡ表达增强的结果；（３）反应性星形胶质化是ＡＳ的自身特性，以ＡＳ肥大为主，ＡＳ受损后，原癌基因ｃ－ｆｏｓ的蛋白参与调节ＡＳ激活的确切机制尚待阐明。

P2X_7受体在原代培养星形胶质细胞生长中的作用

目的探讨ATP是否促进原代培养的星形胶质细胞(astrocytes,As)发生类似反应性星形胶质化的变化以及嘌呤类受体P2X7在该过程中的作用。方法原代As的分离和培养;实时照相观察As形态变化;用Br-dU掺入法及流式细胞观察As增殖变化;用荧光标记实时定量PCR(real-time RT-PCR)检测GFAP mRNA,P2X7mRNA,TGF-β1mRNA表达的变化;用Western blot检测GFAP表达;ELISA检测培养上清中TGF-β1的变化。结果成功分离并培养原代As,免疫荧光鉴定阳性率为99%。不同浓度ATP(50μmol/L,100μmol/L,500μmol/L)作用于As,均可使之表现出类似反应性胶质化的形态改变,表现为胞浆丰富,细胞突起增加且更为明显。ATP100μmol/L作用1d后,As的S期细胞数目明显增加,提示细胞增殖的活跃,5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine,5-BrdU)掺入法显示细胞核阳性率明显增加。500μmol/L浓度的ATP能促进As的GFAP mRNA,P2X7mRNA表达。用P2X7特异性的受体激动剂2′-3-′O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate(BzATP)50μmol/L,75μmol/L,100μmol/L作用于原代培养的As,在培养基有Ca2+情况下可明显促进GFAP表达。在培养基有Ca2+的情况下BzATP作用2h及12h时As中TGF-β1mRNA升高。培养上清中TGF-β1蛋白的含量在8h及48h均有升高。TGF-β1中和抗体能部分抑制P2X7引起的As GFAP含量的增加。结论100μmol/L的ATP可以促进As增殖。各种浓度ATP均可促进As形态产生类似胶质化反应的变化。P2X7特异性受体激动剂BzATP可引发胶质化样反应,并且在有Ca2+的情况下促进TGF-β1的转录和释放。TGF-β1参与了P2X7受体诱导的类似反应性星形胶质化过程。

有机磷农药对星形胶质细胞的影响

有机磷农药是我国生产和使用最多的农药,对神经的损伤是有机磷农药中毒患者致死的主要原因。研究表明星形胶质细胞可能是有机磷农药除乙酰胆碱酯酶之外的作用靶点。有机磷农药能够激活星形胶质细胞,反应性胶质化在有机磷农药的神经毒性中具有两面性,研究者们更倾向于认为它对有机磷农药神经毒性的保护作用。

脊髓损伤后bFGF和GFAP的变化及其意义

目的 研究脊髓损伤后碱性成纤维细胞因子和胶质纤维酸性蛋白的变化及其两者的相关性 ,探讨脊髓损伤后脊髓的自身保护机制以及星形胶质细胞在脊髓损伤中的作用。方法 用免疫组织化学、组织学染色和图象分析的方法检测损伤脊髓碱性成纤维细胞及胶质纤维酸性蛋白表达的动态变化。结果 脊髓损伤后 1d损伤脊髓中碱性成纤维细胞的表达明显增高 ,7d达到高峰 ,14d末回落 ;而胶质纤维酸性蛋白表达则在 1～ 14d内呈进行性增高趋势。两指标相关性显著 (r =0 .777,P =0 .0 0 1)。结论 脊髓损伤后反应性星形细胞胶质化对脊髓的再生和自身修复起着重要作用 ,提示脊髓本身存在自身保护机制。

SOX2/DRD2 signaling pathway facilitates astrocytic dedifferentiation in cerebral ischemic mice

Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.

睫状神经营养因子对新生大鼠培养星形细胞胶质化的影响

在新生大鼠混合培养胶质细胞机械划伤模型上, 研究了睫状神经营养因子(CNTF)对反应性星形细胞胶质化的影响. 损伤后, 在损伤边缘可见典型的星形细胞胶质化过程, 表现为扁平型星形胶质细胞增生肥大, 其胞内GFAP表达增加, O-2A前体细胞向受损区域迁移, 并分化成为突起型星形胶质细胞. 向培养基加入外源性CNTF可促进损伤边缘的扁平型星形胶质细胞的增生及GFAP的表达. 结果提示CNTF可以加强损伤区的星形细胞胶质化.

Effect of ciliary neurotrophic factor on activation of astrocytes in vitro

Objective To observe the activating effect of ciliary neurotrophic factor （CNTF） on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations （in ng/ml： 0, 2, 20, or 200） of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein （GFAP） and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state ceils with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.

Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes

HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.

杂志信息

据《中华妇产科杂志》1997年3月32卷第3期报道 为了了解异位妊娠时子宫内膜的形态变化及其规律,上海医科大学妇产科医院病理科陆惠娟等,收集了经诊断性剖宫病理检查确诊为异位妊娠病例的病理标本共217份(217例),分析其子宫内膜的形态变化,并结合临床资料进行分析。结果:异位妊娠时子宫内膜变化有7种类型: (1)

Protective effects of parecoxib on rat primary astrocytes from oxidative stress induced by hydrogen peroxide

Objective： To investigate the protective effects of parecoxib from oxidative stress induced by hydrogen peroxide （H202） in rat astrocytes in vitro. Methods： All experiments included 4 groups： （1） negative control （NC） group, without any treatment; （2） H202 treatment group, 100 μmol/L H202 treatment for 24 h; （3） and （4） parecoxib pre- treatment groups, 80 and 160 μmol/L parecoxib treatment for 24 h, respectively, and then treated with 100 μmol/L H202. Several indices were investigated, and the expressions of Bax, Bcl-2, and brain-derived neurotrophic factor （BDNF） were quantified. Results： Compared to the NC group, exposure to H202 resulted in significant morphological changes, which could be reversed by pretreatment of parecoxib. In addition, H202 treatment led to loss of viability （P=0.026） and increased intracellular reactive oxygen species （ROS） levels （P〈0.001）, and induced apoptosis （P〈0.01） in the primary astrocytes relative to the NC group. However, in the parecoxib pretreatment groups, all the above changes reversed significantly （P〈0.05） as compared to the H202 treatment group, and were nearly unchanged when compared to the NC group. Mechanical investigation showed that dysregulated Bax, Bcl-2, and BDNF could be im- plicated in these changes. Conclusions： Our results indicated that parecoxib provided a protective effect from oxidative stress induced by exposure to H202.