小鼠胎脑组织活体转基因技术的建立

目的:建立实用的小鼠活体脑组织基因转染技术。方法:将EGFP质粒(CAGS启动子)注射到胎龄16 d(E16d)的胎鼠侧脑室,用镊形电极隔着子宫壁夹住胎鼠头部,在45 V电压下给予5次电脉冲刺激,每次刺激50ms,间隔1 s;转染后不同天数将胎鼠脑组织完整取出,以4%PFA固定后冰冻切片,进行激光共聚焦照相。结果:EGFP质粒被转入小鼠活体脑组织细胞中并获得表达,动物存活率为90%,GFP阳性率高于80%。结论:通过对麻醉剂、电脉冲刺激、质粒浓度、术中术后处理等多种实验条件的摸索,建立了实用的小鼠胎脑组织活体转基因技术。     

扬子鳄胚胎大脑皮层神经元核内包涵体的超微结构特征

孵化40、50d扬子鳄胚胎的海马皮质及刚孵出的小鳄大脑皮层各部的部分神经元细胞核内均有核内包涵体出现。按其形态可将它们分为两大类：膜性包涵体和核体。膜性包涵体位于近内核膜处,直径0．3—0．9μm,是由一到几层膜组成的囊泡状结构;核体体积较小,0．2—0．4μm,位于近核仁处或近内核膜,周缘的电子致密度较高,中心较清亮。本文就扬子鳄的核内包涵体同其它动物的核内包涵体的形态、出现时间进行了比较,并就其作用进行了讨论。     

Sox11基因对发育期小鼠大脑皮层神经元迁移的影响

目的: 以小鼠为实验动物研究精神分裂症易感基因Sox11对皮层神经元迁移的影响。方法: 应用实时荧光定量PCR、原位杂交等技术明确发育期 (E14.5, P0, P7, P14) Sox11于大脑皮层的表达模式;应用质粒构建、转染、胚胎电转、免疫荧光染色等技术,对不同时期 (E17.5, P0, P4, P7) 的小鼠分别转染对照shRNA质粒、mSox11 shRNA质粒和mSox11 shRNA干扰恢复后质粒,研究Sox11在神经元放射性迁移中的作用。结果: 与对照组神经元相比,转染mSox11 shRNA的神经元迁移明显延迟。当对照组神经元有一部分已经到达新皮层的表层时,大部分转染mSox11 shRNA的神经元仍停留在新皮层中间区;使用大鼠Sox11基因 (rSox11) 过表达载体对小鼠Sox11基因的干扰进行恢复后,神经元迁移完成后的分布情况与对照基本一致。小鼠Sox11干扰后和干扰恢复后,室管膜下区 (SVZ)、中间区 (IZ) 和皮层板 (CP) 内迁移神经元分布具有显著性差异 (P＜0.01)。结论: Sox11可以促进皮层神经元的迁移,提示Sox11在小鼠皮层神经元迁移过程中发挥重要功能。     

p85a基因对小鼠大脑皮层投射神经元迁移的影响

Pi3k/Akt信号通路是近年来发现的参与细胞增殖、调控的重要通路,Pi3k激活可介导多种细胞功能,Pi3k由p85a和p110构成。该研究通过干扰p85a基因的表达,探讨其在小鼠大脑皮层投射神经元迁移中的作用。首先,构建p85a基因的对照质粒（scramble）、siRNA质粒（sip85a-1、sip85a-2）和过表达质粒（OEp85a）;接着转染N2a细胞,48 h后,用定量PCR方法检测p85a基因mRNA的表达情况。随后,将sip85a-1、sip85a-2、OEp85a质粒分别转入小鼠大脑,4 d后,借助免疫荧光方法检测皮层神经元的迁移情况。定量PCR结果显示：与对照相比,转染sip85a-1、sip85a-2均能显著降低N2a细胞中p85a基因的mRNA表达,抑制效率约为40%（P〈0.05）;而转染OEp85a质粒后,能显著增加p85a基因mRNA的表达,约为对照组的12倍（P〈0.01）。胚胎电转结果中,各区EGFP阳性神经元数目定量分析显示,sip85a-1、sip85a-2、OEp85a质粒均能显著抑制神经元的迁移（P〈0.05）。大脑发育阶段中,p85a基因在适当范围内,对平衡神经元迁移过程中起着重要作用。     

神经元迁移的细胞和分子机制

在脑的发育过程中,神经元的正确迁移是正常脑组织发生的一个必不可少的环节。在过去的几十年中,通过不同的学科方法,对于神经元迁移的机制有了较好的理解。在细胞水平上,神经元迁移需要3个重复事件的精确调控;在分子水平,与神经元迁移相关的胞外信号分子已经被鉴定,而且大量的胞内信号通路也已经被阐明。     

我科研人员关于大脑皮层神经元放射状迁移导向机制的最新研究成果在Nature Neuroscience上发表

2008年1月10日,Nature Neuroscience发表了中科院上海生科院神经所袁小兵研究组关于大脑皮层神经元放射状迁移导向机制的最新研究成果。这些研究首次证明了神经元的放射状迁移受胞外导向分子的指引。     

体外培养的鸡胚神经元迁移的光镜和扫描电镜的研究

用鸡胚神经细胞为材料,建立神经细胞体外培养技术,相差显微镜观察到鸡胚神经元可以沿着神经胶质细胞纤维运动。扫描电子显微镜揭示体外培养神经元与胶质细胞的关系。     

刺激大脑皮层体感Ⅰ区和大脑脚对大鼠脊髓背角神经元伤害感受性反应的影响

在大鼠用玻璃微电极细胞外记录的方法,观察了刺激皮层体感Ⅰ区(SI区)和大脑脚(CP)对皮肤强电刺激诱发的脊髓背角广动力范围(WDR)神经元长潜伏期反应(C-反应)的影响。结果表明刺激SI区对背角WDR神经元C-反应的影响以抑制为主,刺激CP的作用与刺激SI区的作用相似,但刺激CP更为有效。抑制作用的持续时间在不同神经元差别很大,短者在刺激停止后仅持续400ms,长者可达10min以上。静注纳洛酮对抑制作用无明显影响,静注二甲麦角新碱在部分神经元可使抑制作用明显减弱或完全消失,提示5-HT部分参与皮层下行抑制作用的实现,而内鸦片肽则否。     

miR-346通过靶向调控SRSF10抑制胶质瘤细胞迁移、侵袭和增殖

胶质瘤是最常见的肿瘤之一,预后较差。有研究表明,microRNAs(miRNAs)与包括胶质瘤在内的多种肿瘤有密切联系。然而,miR-346通过靶向调控丝氨酸/精氨酸剪接因子10(serine/arginine splicing factor 10,SRSF10)抑制胶质瘤细胞迁移、侵袭和增殖能力的分子机制目前仍不明确。对GSE165137与GSE139031芯片进行差异表达分析结果显示,miR-346在两个数据集中共同下调,运用CGGA数据库分析显示,miR-346高表达的患者总生存期明显提高(P<0.0001),miR-346的表达随胶质瘤WHO分级升高而降低(P<0.05)。实时荧光定量PCR(qRT-PCR)结果显示,miR-346在胶质瘤细胞U251中的表达显著降低(P<0.05),且miR-346过表达质粒转染成功(P<0.05)。Transwell实验,5-乙炔基-2脱氧尿嘧啶核苷(EdU)增殖实验和细胞克隆形成结果显示,与对照组相比,过表达miR-346后U251细胞的迁移、侵袭和增殖能力下降(P<0.05)。生物信息学方法预测miR-34...     

HDAC6通过调控HSP90影响Aβ诱导的大鼠海马神经元细胞功能和形态的研究

该文旨在探讨组蛋白去乙酰化酶6(histone deacetytase 6,HDAC6)通过调控热休克蛋白90(heat shock protein 90,HSP90)影响体外培养Aβ诱导的大鼠海马神经元细胞功能及形态的变化。取孕18天SD大鼠胎鼠,体外培养海马神经元细胞,7天后用β-tubulin III抗体鉴定海马神经元纯度。用寡聚体Aβ1-42干预24 h,随后分别加入HDAC6抑制剂TSA、HDAC6激动剂Theo、HSP90抑制剂Gane或生理盐水;细胞不加Aβ1-42干预作为对照。用CCK8法检测细胞的活力,免疫荧光法观察神经元细胞突起的长度及形态变化,Western blot检测HDAC6和HSP90蛋白表达,qRT-PCR检测hsp90基因的mRNA表达水平。结果表明,应用HDAC6抑制剂可下调HDAC6水平,同时促进了hsp90 mRNA和HSP90蛋白的表达,也提高了海马神经元活性、突起长度和分支数目,HDAC6激动剂则引起相反的效应;而应用HSP90抑制剂后则降低了神经元活性和突起长度以及分支数目,但HDAC6没有变化。因此,推测HDAC6可能通过调控HSP90水平影响Aβ诱导的大鼠海马神经元细胞功能和形态的变化。     

Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum

We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.     

Maturation of Cell-Substratum Focal Adhesions Induced by Depolymerization of Microtubules is Mediated by Increased Cortical Tension

Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA.

Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA.

Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.     

Gene application with in utero electroporation in mouse embryonic brain

Mouse genetic manipulations, such as the production of gene knock-out, knock-in, and transgenic mice, have provided excellent systems for analysis of numerous genes functioning during development. Nevertheless, the lack of specific promoters and enhancers that control gene expression in specific regions and at specific times, limits usage of these techniques. However, progress in in utero systems of electroporation into mouse embryos has opened a new window, permitting new approaches to answering important questions. Simple injection of plasmid DNA solution and application of electrical current to mouse embryos results in transient area- and time-dependent transfection. Further modification of the technique, arising from variations in types of electrodes used, has made it possible to control the relative size of the region of transfection, which can vary from a few cells to entire tissues. Thus, this technique is a powerful means not only of characterizing gene function in various settings, but also of tracing the migratory routes of cells, due to its high efficiency and the localization of gene expression it yields. We summarize here some of the potential uses and advantages of this technique for developmental neuroscience research.     

Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP

Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.     

Effects of Jarastatin, a Novel Snake Venom Disintegrin, on Neutrophil Migration and Actin Cytoskeleton Dynamics

A new disintegrin, an RGD-containing peptide of 6 kDa called jarastatin, was purified from Bothrops jararaca venom. It is a potent inhibitor of platelet aggregation induced by ADP, collagen, and thrombin. The effect of jarastatin on neutrophil migration in vivo and in vitro and on the actin cytoskeleton dynamics of these cells was investigated. Incubation in vitro with jarastatin significantly inhibited, in a concentration-dependent manner, the chemotaxis of human neutrophils toward fMLP, IL-8, and jarastatin itself. Despite this inhibitory effect, jarastatin induced neutrophil chemotaxis. A significant increase of F-actin content was observed in jarastatin-treated neutrophils. Furthermore, as demonstrated by confocal microscopy after FITC-phalloidin labeling, these cells accumulated F-actin at the plasmalemma, a distribution similar to that observed in fMLP-stimulated cells. Pretreatment of mice with jarastatin inhibited neutrophil migration into peritoneal cavities induced by carrageenan injection. The results suggest that binding of jarastatin to neutrophil integrins promotes cellular activation and triggers a dynamic alteration of the actin filament system and that this is one of the first event in integrin-mediated signaling.