巢蛋白的克隆、表达、抗体制备及免疫组化分析

目的 在原核系统中克隆、表达并纯化巢蛋白（nestin）．制备特异性nestin抗体,用于研究nestin在中枢神经系统发育中的生物学特性．探索神经发育及再生规律。方法 采用RT—PCR方法由人神经干细胞中获取nestin cDNA,与原核表达载体pQE30连接,构建重组载体pQE30-nestin。测序后．将重组质粒转入大肠杆菌M15,IPTG诱导表达His融合蛋白。Ni-NTA亲和层析柱纯化后,SDS-PAGE电泳和Westem blotting鉴定。用此蛋白免疫BALB／c小鼠,获得抗血清。Westemblotting、ELISA和免疫组织化学分析鉴定获取的抗血清。结果 成功由人神经干细胞中克隆nestin cDNA片段。胛G诱导重组质粒pQE30-nestin表达出一相对分子质量约25000的His融合蛋白并被Ni．NTA成功纯化．Westem blotting证明其确为nestin蛋白。动物免疫后．经Westem blotting、ELISA和免疫组化鉴定,获得的抗血清可特异性结合重组nestin蛋白、发育期人及大鼠脑内的nestin蛋白。结论 获得了重组人nestin蛋白．制备的抗血清不仅可识别重组nestin蛋白,亦可识别人及大鼠脑组织内的nestin蛋白。     

戊四氮点燃大鼠海马星形胶质细胞巢蛋白的表达

目的：观察戊四氮点燃大鼠海马各区巢蛋白(nestin)的表达,以探讨其与癫痫发病机制的关系。方法：将20只成年雄性SD大鼠随机分为实验组和对照组,实验组采用戊四氮点燃大鼠模型,观察大鼠痫性发作行为和脑电图表现,用免疫组织化学方法观察海马各区星形细胞活化标志巢蛋白表达的变化。结果：实验组大鼠于4周后均达到点燃状态,而对照组行为正常。2组脑电图背境电活动以a节律为主,实验组Ⅳ或Ⅴ级痫性发作时脑电图均出现典型的高波幅棘慢波、尖慢波。实验组海马各区nestin阳性星形细胞较对照组明显增多。结论：戊四氮点燃引起海马内nestin阳性星性细胞明显增加,提示癫痫脑组织内存在星形细胞增生和活化,这可能是癫痫脑组织胶质化及点燃维持的病理基础。     

叶酸对胎鼠神经干细胞体外增殖的影响

目的探讨叶酸对体外培养的胚胎大鼠神经干细胞(neural stem cells,NSCs)增殖的影响。方法本研究采用显微解剖、机械吹打、无血清悬浮培养方法分离培养鼠胚大脑NSCs,用巢蛋白(nestin)免疫荧光染色对其进行鉴定,5'-溴脱氧尿嘧啶(BrdU)掺入法检测细胞增殖状态;设立正常对照组,叶酸低、高剂量(培养液中添加叶酸4.0 40 mg/L)和叶酸缺乏组(培养液中添加叶酸拮抗剂甲氨蝶呤0.4mg/L);通过nestin+BrdU免疫荧光双标记和绘制细胞生长曲线(MTT法)检测叶酸对细胞增殖状态的影响。结果无血清培养液中可形成大量呈nestin抗原阳性细胞组成的神经球,BrdU免疫标记证实培养的NSCs具有增殖能力。免疫荧光双标记和MTT结果显示,两个叶酸干预组NSCs增殖能力较对照组显著增强。结论体外培养的胚胎大鼠NSCs具有增殖和自我更新的能力;叶酸可以在一定程度上增强NSCs的增殖能力,促进神经干细胞的生长。     

脑衍生神经营养因子对胚胎神经干细胞神经细胞黏附分子的表达

目的：探讨脑源性神经营养因子（BDNF）对胚胎神经干细胞神经细胞黏附分子（NCAM）表达的影响。方法：体外培养和鉴定SD大鼠海马胚胎神经干细胞,从而获得神经干细胞系。实验分为BDNF组和对照组,分别在培养后12 h和24 h对神经干细胞NCAM的表达进行免疫细胞组织化学检测,图象分析NCAM阳性个数、胞体面积、细胞周长。结果：来源于胚胎的神经干细胞增殖能力较强且Nestin阳性,神经干细胞能够表达NCAM,且BDNF组高于对照组,进一步观察发现BDNF组NCAM的各项参数值在24 h明显高于12 h（P〈0.05）。结论：BDNF能促进NCAM在神经干细胞中的表达。     

Culturing of Specialized Glial Cells (Olfactory Ensheathing Cells) of Human Olfactory Epithelium

A monolayer of dissociated glial cells of human olfactory epithelium was cultured in Petri dishes and 12-well plates using a polylysine-laminin substrate. Primary cultures were sub-cultured after 10–15 days. The cell cultures were analyzed by phase contrast microscopy at all stages of culturing. A cytological study involved histological methods (trypan blue staining) and immunocytochemical visualization of GFAP, nestin, and low-affinity nerve growth factor receptors. At the final stage of culturing (5 passages) the monolayer cultures included 2 types of cells: GFAP- and p75-positive glial cells and nestin-positive fibroblasts.__________Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 95–98, 2005     

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.     

NMDA receptor antagonist treatment induces a long-lasting increase in the number of proliferating cells, PSA-NCAM-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus

During adulthood, neural precursors located in the subgranular zone of the dentate gyrus continue to proliferate, leading to the generation of new granule neurons. These recently generated cells transiently express the polysialylated form of the neural cell adhesion molecule, PSA-NCAM, and are supported by radial glia-like cells that are likely to play a role in neuronal migration and differentiation, or even act as their precursors. Previous reports indicate that treatment with NMDA receptor antagonists stimulates adult neurogenesis in the dentate gyrus, and because of the potential therapeutic value of this approach, we were interested in further characterizing the consequences of pharmacologically modulating this process. We treated adult rats with the competitive NMDA receptor antagonist, CGP43487, and examined cell proliferation, PSA-NCAM expression, and changes in the radial glia cell population in the subgranular zone at different time points. In addition, we sought to determine if this treatment led to changes in cell death or gliotic reactions. The number of proliferating cells in the subgranular region of the dentate gyrus was increased significantly 2 days after treatment and it remained elevated 7 days postinjection. PSA-NCAM-immunoreactive granule cells and nestin-expressing radial glia-like cells also increased in number 7 days after the treatment. In contrast, we did not observe any change in granule cell death, and we were unable to detect any microglial or astroglial reaction during the first 7 days after treatment. Thus, NMDA receptor antagonist treatment serves as a valuable tool to increase neurogenesis in the adult hippocampus without undesirable collateral deleterious effects.     

缺血再灌注损伤大鼠脑内神经巢蛋白的变化及通心络对它的影响

目的 探讨神经巢蛋白（nestin）在脑缺血再灌注损伤后神经细胞的活化增殖情况及通心络对其影响。方法 采用大鼠缺血再灌注损伤（MCAO）模型，应用免疫组织化学方法观察缺血后3、7、14以及21d缺血侧室管膜及室管膜下区（SVZ）、海马齿状回（HDG）神经巢蛋白的变化。给予模型大鼠通心络灌胃，观察神经干细胞增殖分化的变化。结果 神经巢蛋白阳性细胞随缺血再灌注时间的延长，荧光强度值增加，第7、14、21天组与假手术组比较，差异具有显著性（P〈0．05）。造模后通心络组BrdU阳性细胞荧光强度值和BrdU＋nestin免疫双标荧光强度值均高于脑缺血再灌注模型组,差异显著（P〈0．05）。结论 大鼠缺血再灌注损伤后可引起其缺血侧SVZ、HDG区神经干细胞反应和增殖；而通心络可显著增加MCAO大鼠神经干细胞增殖分化能力。     

Increase of nestin-immunoreactive neural precursor cells in the dentate gyrus of pediatric patients with early-onset temporal lobe epilepsy.

A considerable potential for neurogenesis has been identified in the epileptic rat hippocampus. Here, we explore this feature in human patients suffering from chronic mesial temporal lobe epilepsy. Immunohistochemical detection of the neurodevelopmental antigen nestin was used to detect neural precursor cells, and cell-type specific markers were employed to study their histogenetic origin and potential for neuronal or glial differentiation. The ontogenetic regulation of nestin-positive precursors was established in human control brains (week 19 of gestation-15 years of age). A striking increase of nestin-immunoreactive cells within the hilus and dentate gyrus could be observed in a group of young patients with temporal lobe epilepsy (TLE) and surgical treatment before age 2 years compared to adult TLE patients and controls. The cellular morphology and regional distribution closely resembled nestin-immunoreactive granule-cell progenitors transiently expressed during prenatal human hippocampus development. An increased Ki-67 proliferation index and clusters of supragranular nestin-immunoreactive cells within the molecular layer of the dentate gyrus were also noted in the group of young TLE patients. Confocal studies revealed colocalization of nestin and the betaIII isoform of tubulin, indicating a neuronal fate for some of these cells. Vimentin was consistently expressed in nestin-immunoreactive cells, whereas cell lineage-specific markers, i.e., glial fibrillary acidic protein, MAP2, neurofilament protein, NeuN, or calbindin D-28k failed to colocalize. These findings provide evidence for increased neurogenesis in pediatric patients with early onset of temporal lobe epilepsy and/or point towards a delay in hippocampal maturation in a subgroup of patients with TLE.