体外培养的神经球中表达神经巢蛋白细胞的流式细胞术检测

采用体外培养不同时间的神经干细胞球 ,间接荧光法标记神经巢蛋白 (nestin) ,以流式细胞术检测神经干细胞球中表达nestin细胞的情况。结果显示 :在培养不同时间的细胞群体中均检出 nestin阳性细胞 ,培养 7d、1月、3月和 6月的细胞群含nestin阳性细胞数百分比分别为 (4 2 .2± 7.6) %、(4 1.7± 7.3 ) %、(3 3 .8± 12 .9) %和 (3 7.1± 5 .0 ) % ,几个时间点的细胞群体所含的 nestin阳性细胞数的百分比之间无显著性差异。提示 ,在体外培养条件下 ,神经干细胞群体可能通过自我更新和分化的调控 ,保持 nestin阳性细胞于一种较为恒定的比例。     

Intracranial Injury Acutely Induces the Expression of the Secreted Isoform of the CNS-Specific Hyaluronan-Binding Protein BEHAB/Brevican

Hyaluronan (HA) plays an important role in tissue reorganization in response to injury. The mechanisms by which HA participates in these processes are likely to include HA-binding proteins. Previously, we reported the cloning and initial characterization of a central nervous system (CNS)-specific HA-binding protein, BEHAB (brain enriched hyaluronan binding), which was independently cloned in another laboratory and named brevican. BEHAB/brevican mRNA is expressed in the ventricular zone coincident with the initial proliferation and migration of glial cells and in surgical samples of human glioma, where glial-derived cells proliferate and migrate. To determine whether BEHAB/brevican is also expressed during the cellular proliferation and migration associated with CNS injury, we have examined BEHAB/brevican expression during reactive gliosis. BEHAB/brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol (GPI)-anchored, isoforms. The secreted, but not the GPI-anchored, isoform is up-regulated in response to a stab wound to the adult rat brain. The temporal regulation and spatial distribution of BEHAB/brevican expression parallel the gliotic response and the expression of the intermediate filament protein nestin. The up-regulation of BEHAB/brevican in response to CNS injury suggests a role for this extracellular matrix molecule in reactive gliosis. Glial process extension, a central element in the glial response to injury, may require the reexpression of both cytoskeletal and matrix elements that are normally expressed during the glial motility seen in the immature brain.     

Expression and regulation of kainate and AMPA receptors in the rat neural tube

We analyzed the expression and regulation of glutamate receptor subunits in the rat neural tube (10 day embryos) and in cell cultures derived from this tissue. In the cultures, all cells were stained with antibodies against the neural progenitor marker nestin. More than 50% of the cells were also stained by the monoclonal antibodies LB1 or A2B5, which bind to neuronal and glial progenitors. Approximately 6% of the cells were stained with antibodies for the low affinity NGF receptor, a neural crest cell marker. A small percentage of cells differentiated to neurons or astrocytes, as determined by staining with anti-neurofilament and anti-GFAP antibodies, respectively. RT-PCR analysis of neural tube tissue and culture mRNAs demonstrated that the AMPA receptor subunits GluR3 and 4 and the kainate receptor subunits GluR6, 7, KA1 and KA2 were detectable at E10. The kainate receptor subunits GluR6 and KA2 were upregulated by culture conditions which stimulated cell differentiation, as determined by concomitant downregulation of nestin mRNA. Both in neural tube tissue and in cultured cells, GluR6 was 100% unedited. Finally, both GluR6 and KA2 proteins could be detected in subpopulations of neural progenitors and differentiated neurons. Our data indicate that kainate receptor genes are expressed in undifferentiated progenitor cells of the neural tube at E10, and are upregulated during neural cell differentiation. J. Neurosci. Res. 52:356–368, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Clinico‐pathological feature of pilomyxoid astrocytomas: Three case reports

Pilomyxoid astrocytoma (PMA) is a newly identified variant of pilocytic astrocytoma (PA). We report three cases of PMA with comparison to seven cases of PA in terms of their clinicopathological features. The three cases occurred at the ages of 2, 36 and 6 years, and their tumors were located in the left basal ganglia, the pineal gland, and the cerebellum, respectively. They were diagnosed PMA by surgical specimens that showed a characteristic monomorphous architecture with an angiocentric growth pattern and myxoid background. One patient developed localized relapse at 6 months after the surgery, but the other patients remained alive without tumor progression more than 5 years after treatment. In analysis of the immunohistochemical association in PMA and PA, no specific staining was found to be useful for differential diagnosis of PMA from PA. The expression of biomarkers including O‐6‐methylguanine‐DNA methyltransferase, p53, MIB‐1, and EGF receptor neither distinguished PMA from PA nor correlated with outcome. But almost all PMA and PA that demonstrated prominent positivity for nestin showed a high MIB‐1 labelling index (LI), and four of these five patients suffered a relapse in the early phase. These results suggest that immunohistochemical expression of nestin and MIB‐1 LI may correlate with the aggressiveness of the tumor in PA and PMA.     

Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice

Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice 3 days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post‐injury (DPI). BrdU‐labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all timepoints. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations that could be targeted by interventions. J. Comp. Neurol. 518:1370–1390, 2010. © 2009 Wiley‐Liss, Inc.     

Expression and distribution patterns of the stem cell marker,nestin, and the stem cell renewal factor,BMI-1, in normal human nasal mucosa and nasal polyps

Conclusions: These results suggest that nestin and BMI-1 are candidates for stem cell markers and renewal factors in human nasal mucosa, may contribute to tissue homeostasis and differentiation in the epithelium and submucosal glands of normal nasal mucosa, and may play a role in proliferation of nasal polyps. Objectives: The stem cell marker, nestin, and the stem cell renewal factor, BMI-1, have been identified in a variety of inflammatory and normal tissues, implicating these markers in tissue regeneration. Materials and methods: We investigated the expression and distribution of nestin and BMI-1 in normal nasal mucosa and nasal polyps, using RT-PCR, immunohistochemistry, and Western blotting. Results: Nestin and BMI-1 were localized to the epithelium and submucosal glands of normal nasal mucosa and nasal polyps. The expression of nestin was confined to the plasma membrane and cytoplasm, whereas BMI-1 showed a nuclear staining pattern. In normal nasal mucosa and nasal polyps, nestin and BMI-1 expression was strongest in the basal portion of the epithelial layer, and decreased toward the upper portion. In the submucosal glands, weak to strong expression was commonly detected in the glandular acini. There was no significant difference in the level of expression of nestin and BMI-1 between normal nasal mucosa and nasal polyps.     

骨髓间充质干细胞向神经元样细胞分化前后基因及Ca2+浓度的变化

背景：单唾液酸四己糖神经节苷脂(Monosialotetrahexosylganglioside,GM1)在神经细胞的生长发育、分化、再生和细胞内外信息传递等多种生理过程中发挥重要作用。 目的：探讨GM1对骨髓间充质干细胞按照Woodbury经典诱导方案将其诱导分化为神经元样细胞前后基因方面和细胞内游离Ca2+浓度的变化。 方法：分离纯化SD大鼠骨髓间充质干细胞进行传代培养,传至第5代时,待细胞融汇成致密单层后,加入50 mmol/L神经节苷脂设为GM1组,预培养24 h后按照Woodbury经典诱导方案进行诱导,设置对照组,采用免疫组化和Realtime PCR技术分别检测诱导前后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白的蛋白和mRNA表达的变化,采用激光扫描共聚焦显微镜检测诱导前后细胞内游离钙离子浓度的变化。 结果与结论：①加入GM1组诱导分化后较对照组生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白表达水平增高(P < 0.05),GM1能够促骨髓间充质干细胞诱导分化为神经元样细胞。②更换诱导液后,两组细胞内荧光强度逐渐增加,到100 s 达高峰值,其后逐渐减弱,但20 min 时细胞荧光强度仍高于诱导前,加入GM1组,细胞内游离Ca2+浓度较对照组有所增加(P < 0.05)。说明GM1能够促进细胞内Ca2+浓度增加,游离Ca2+在诱导过程中可能有促进作用。③诱导后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白基因表达改变不显著,说明Woodbury经典诱导方案可能为转录后水平即蛋白水平的调控。     

神经干细胞移植治疗大鼠局灶性脑缺血损伤

背景：近年来,神经干细胞移植已成为治疗神经退行性疾病和中枢神经系统损伤的研究热点。 目的：探讨神经干细的定向分化调控机制和神经干细胞移植治疗大鼠局灶性脑缺血损伤的研究进展。 方法：以“neural stem cells, stem cell transplantation, ischemic brain injury”为检索词,检索Pubmed数据库1990至2012年相关文献;以“神经干细胞,干细胞移植,缺血性脑损伤”为检索词,检索CNKI数据库2005至2012 年相关文献。分析神经干细的定向分化调控机制和神经干细胞移植治疗大鼠局灶性脑缺血损伤的内容,排除重复研究。 结果与结论：①体外分离培养的神经干细胞有胚胎来源、脐血来源和成体来源,主要采用机械分离法和胰酶消化法进行分离。②目前体外培养的神经干细胞分离鉴定的标记物有巢蛋白、波形蛋白1、5-溴脱氧尿嘧啶核苷、神经元特异性烯醇化酶等。③神经干细胞的分化调节是通过正负双重作用实现的,负性调节是通过对称性的分裂来增加神经干细胞数量,包括Notch信号途径和一些生长因子等。正性调节诱导神经干细胞分化,包括参与细胞合成的骨形态发生蛋白信号途径等。④神经干细胞移植的时间窗选择在实验动物脑缺血两三周后,时间过早和过晚均不适合细胞的存活。神经干细胞通过脑立体定位仪直接进行脑内移植治疗大鼠局灶性脑缺血损伤,移植后可见细胞在局灶性脑缺血大鼠脑室内和梗死中心均可长期存活,并可广泛迁移,移植神经干细胞后观察到其运动行为学评分有明显提高。缺血性脑卒中的神经干细胞移植治疗还存在一些问题需要解决,未来的临床应用前景广阔,是缺血性脑卒中患者的新希望。     

微管相关蛋白2和巢蛋白在人胚胎胃组织中的表达

摘要：目的探讨微管相关蛋白2 (MAP-2)和巢蛋白(Nestin) 与人胚胎胃组织的发育关系。方法应用免疫组织化学PV法检测第
2、3、4三个月龄段,人胚胎胃壁、贲门和幽门组织内MAP-2和Nestin的表达规律。结果第2、3、4三个月龄段,MAP-2和Nestin阳
性表达主要集中于人胚胎胃壁、贲门和幽门组织黏膜下神经丛和肌间神经丛,随胎龄的增大,MAP-2和Nestin蛋白在肌间神经
丛内阳性表达细胞数量和强度均呈增高趋势。而MAP-2和Nestin蛋白在胃壁、贲门和幽门组织的黏膜上皮和腺体处均呈阴性
表达。结论MAP-2和Nestin参与调节人胚胎胃组织的生长发育过程。