睫状神经营养因子对体外培养星形胶质细胞的激活作用

目的 观察睫状神经营养因子(CNIF)对体外培养星形胶质细胞的细胞激活作用。方法分别给予不同浓度(0、2、20、200ng／ml)的CNTF孵育有血清培养和无血清培养的星形胶质细胞,采用免疫细胞化学技术及流式细胞术,观察星形胶质细胞形态及细胞周期的变化。结果有血清培养和无血清培养时CNTF均使星形胶质细胞GFAP表达增强,胞核肥大。有血清培养时CNTF还可以促进星形胶质细胞进入细胞周期进行增殖;无血清培养时CNTF无此效应。结论无血清培养时CNTF可以刺激星形胶质细胞进入活化状态,但不刺激其增殖;有血清培养时CNTF可以协助血清中的丝裂原引起星形胶质细胞增殖。     

成年大鼠嗅鞘细胞与嗅觉神经成纤维细胞的原代培养及鉴定

目的 建立一种原代提取嗅鞘细胞与嗅觉神经成纤维细胞混合培养的方法.方法 自2.5月龄SD大鼠嗅球最外两层分离嗅鞘细胞和嗅觉神经成纤维细胞进行混合培养,并不进行纯化,分别于7 d、10 d、14 d行免疫细胞化学鉴定,并计算各个时间点嗅鞘细胞的纯度.结果 体外培养的嗅鞘细胞主要呈两极或多极状,而嗅觉神经成纤维细胞则成扁平的像成纤维细胞的形态,免疫细胞化学结果显示嗅鞘细胞呈p75 NGFR阳性,嗅觉神经成纤维细胞呈fibronectin阳性,两种细胞都呈vimentin阳性,在7 d、10 d、14 d各个时间点嗅鞘细胞分别占混合培养的34.1%、25.6%、8.6%.结论 从成年大鼠嗅球最外两层分离的培养中主要包含嗅鞘细胞和嗅觉神经成纤维细胞,嗅鞘细胞在混合培养中所占的比例随培养时间的延长而逐渐降低.     

神经妥乐平体外诱导大鼠骨髓基质细胞分化为神经元样细胞

目的探索神经妥乐平(NT)体外诱导大鼠骨髓基质细胞(bone marrow stromal cells,rMSCs)分化为神经元样细胞的可行性,以期为临床应用MSCs治疗神经系统疾病奠定基础。方法取一月龄SD大鼠骨髓,分离出MSCs进行培养、扩增、纯化。用NT诱导MSCs分化为神经元样细胞。用神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)免疫细胞化学染色鉴定阳性细胞。结果MSCs经诱导后胞体变圆,伸出细长突起,呈神经元样形态。免疫组化鉴定显示(31.50±7.32)%的细胞表达NSE阳性,(45.30±9.38)%的细胞表达GFAP阳性。结论MSCs在体外可被NT诱导分化为神经元样细胞。     

大鼠脑皮质星形胶质细胞的限制性细胞培养

介绍一种新的脑组织星形胶质细胞培养方法即限制性细胞培养（constraint cell culture）。常规分离纯化星形胶质细胞,将其低密度种植,维持在添中低量血清的化学成分限定的培养基中培养,并在长时期内不给予更换或补加培养液。利用波形蛋白(vimentin）和胶质纤维酸性蛋白（glial fibrary acidic protein）抗体的免疫荧光染色法鉴定观察不同培养时期的星形胶质细胞及其形态学变化。结果发现星形胶质细胞在最初的5天之内有一定程度的增殖,未出现过度增殖导致的细胞相互融合现象;接下来的3－5天内细胞形态明显分化,星形胶质细胞突起细长、胞体明显缩小、形态多样,最后细胞突起之间相互连接形成星形胶质细胞网络,并在相当长的时间内保持不变。实验结果显示在限制细胞种植密度和限制给予培养液的培养条件下星形质细胞的体外形态发育与在体的情形基本一致。提示该细胞培养方法可能有助于研究中枢神经系统中星形胶质细胞的生理功能。     

IL-1β和Glu联合应用对体外培养的新生大鼠大脑皮质星形胶质细胞周期的影响

目的研究IL-1β(interleukin-1 beta) 单独应用及与谷氨酸(Gluamate,Glu)联合应用对体外纯化培养的大鼠大脑皮质星形胶质细胞细胞周期的影响.方法将纯化培养的星形胶质细胞血清剥夺培养24 h后,(1)加入不同浓度(0、10、100、1000 U/ml)的IL-1β;(2)加入浓度100U/ml的IL-1β分别作用24、48、72 h;(3)加入浓度100U/ml IL-1β 1mmol/L Glu分别作用24、48、72 h; 采用流式细胞术观察星形胶质细胞周期的变化.结果 (1)不同浓度的IL-1β可使星形胶质细胞S和G2/M期的细胞指数较对照组增高,在一定范围内随着IL-1β浓度的增加,星形胶质细胞的增殖更明显,同一浓度的IL-1β其作用随着时间的延长而逐渐衰减,(2)IL-1β与Glu联合应用较IL-1β单独应用星形胶质细胞的增殖更明显.结论 IL-1β激活星形胶质细胞,并启动细胞周期进程,促使星形胶质细胞增殖;IL-1β与Glu在诱导星形胶质细胞增殖时有一定的协同作用.     

胰蛋白酶消化强度优化获得高纯度体外培养的星形胶质细胞

本文旨在观察胰蛋白酶消化对体外培养的星形胶质细胞纯度的影响,优化星形胶质细胞培养方法。常规分离新生Sprague Dawley(SD)大鼠大脑皮质,分别用0.25%胰蛋白酶消化20、30和40 min制备单细胞悬液并接种细胞。细胞长满瓶底时进行恒温摇床振荡,前两个不同消化时间组再分为常规消化的对照组和二次胰蛋白酶消化组进行传代纯化。倒置相差显微镜观察细胞生长情况,MTT法检测细胞增殖,GFAP免疫荧光分析星形胶质细胞纯度并观察其形态,流式细胞术分析细胞凋亡。结果显示,胰蛋白酶消化20 min的细胞在原代培养9 d长满瓶底。而延长胰蛋白酶消化时间至30 min,细胞增殖更快,培养7 d即可铺满瓶底,且星形胶质细胞形态正常,纯度达(70.2±4.0)%,较20 min组有显著性提高(P0.05)。40 min组虽然星形胶质细胞纯度也较20 min组有所提高,但细胞增殖缓慢且损伤明显。在恒温摇床振荡结束后,进行二次胰蛋白酶消化可减少传代后杂细胞数量。二次胰蛋白酶消化组第一代(P1)的GFAP阳性率普遍高于各自对照组,其中30 min+二次胰蛋白酶消化组GFAP阳性率为(98.1±1.7)%,相当于20 min+对照组P3水平,且二者的凋亡率无显著性差异。以上结果表明,胰蛋白酶消化30 min+二次消化能有效提高星形胶质细胞纯度,缩短原代培养及纯化时间,是体外快速获得高纯度星形胶质细胞的有效方法。     

恒河猴大脑皮质星形胶质细胞的原代培养及鉴定

为建立有效纯度高的恒河猴星形胶质细胞体外培养体系,无菌条件下取6月龄恒河猴婴猴的大脑皮质,除去白质,充分剪碎后机械吹打制成细胞悬液接种培养,待原代培养的细胞长满培养皿后,通过恒温摇床振荡和传代差速贴壁除去寡突胶质细胞、成纤维细胞等,得到纯化后的星形胶质细胞,并用GFAP-FITC(glial fibrillary acidic protein-fluorescein isothiocyanate)免疫荧光法对所获细胞进行鉴定。分离培养的细胞具备典型的星形胶质细胞形态,并表达星形胶质细胞特异性抗原GFAP(glial fibrillary acidic protein),纯化后获得了高纯度的恒河猴星形胶质细胞。采用该培养方法成功建立原代恒河猴星形胶质细胞培养体系,为体外研究嗜神经性病毒、神经系统疾病及其相关中枢神经病理机制等提供了可靠的细胞模型。     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

成年转基因小鼠嗅鞘细胞的培养、纯化及生物学特性

已有多项研究表明,嗅鞘细胞具有修复中枢及外周神经损伤的潜能。我们选用了表达增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,eGFP)的成年小鼠,分离其双侧嗅球嗅神经纤维层及嗅小球层细胞,体外原代培养并予以纯化。同时结合共聚焦、相差显微镜,细胞增殖分析及免疫组织化学鉴定等技术,对其生物学活性进行研究。结果表明:(1)原代培养转基因成年小鼠嗅球嗅鞘细胞(Olfactoryensheathingcells,OECs)15d后,主要存在两种不同形态和免疫组织化学特征的细胞。一种是带有长突起的双极或多极OECs,表达P75~(NIR)(P75lowaffinityneurotrophicreceptor)S100和胶质原纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)。另一种则是对Thy1.1抗体免疫反应阳性,呈扁平或内皮样形态的成纤维细胞。(2)根据不同类型细胞在未覆层的培养器皿上贴壁速度的差异,我们建立了一种简单易行、不需任何抗体或昂贵仪器的细胞纯化方法,获得了大量高纯度的OECs。(3)在连续纯化培养22d后,OECs仍能保持较高的增殖活性。本实验支持和丰富了OECs发育的相关理论,为进一步体内移植修复CNS损伤提供了理想的材料。     

胶质细胞生长因子的研究进展

胶质细胞生长因子(glial growth factor,GGF)是neuregulin基因的产物。GGF与erbB受体的异二聚体或同二聚体结合,催化多肽链中的酪氨酸磷酸化,激活下游信号分子而发挥其生理作用。GGF及其受体在发育及成熟神经系统中广泛分布。GGF限定神经嵴细胞,使其向雪旺氏细胞分化,并在雷旺氏细胞发育过程中发挥重要作用。GGF能够刺激少突胶质细胞前体细胞、少突胶质细胞和星形胶质细胞增殖,抑制少突胶质细胞前体细胞分化成少突胶质细胞,抑制O-2A细胞分化成星形胶质细胞。GGF能够促进神经元沿着放射状的胶质细胞迁移,促进培养的视网膜神经元存活和突触生长。     

RhoA-ROCK-Myosin pathway regulates morphological plasticity of cultured olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are glial cells in the olfactory system with morphological and functional plasticity. Cultured OECs have the flattened and process-bearing shape. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of their morphological plasticity remains elusive. Using RhoA FRET biosensor, we found that the active RhoA signal mainly distributed in the lamellipodia and/or filopodia of OECs. Local disruption of these active RhoA distributions led to the morphological change from the flattened into process-bearing shape and promoted process outgrowth. Furthermore, RhoA pathway inhibitors, Toxin-B, C3, Y-27632 or over-expression of DN-RhoA blocked serum-induced morphological change of OECs from the process-bearing into flattened shape, whereas the activation of RhoA pathway by lysophosphatidic acid (LPA) promoted the morphological change from the process-bearing into flattened shape. Finally, ROCK–Myosin–F-actin as a downstream of RhoA pathway was involved in morphological plasticity of OECs. Taken together, these results suggest that RhoA–ROCK–Myosin pathway mediates the morphological plasticity of cultured OECs in response to extracellular cues.     

Axonal signalling and the making of olfactory ensheathing cells: a hypothesis

Olfactory ensheathing cells (OECs) are Schwann cell-like glial cells of the olfactory system that promote neural repair under experimental conditions. It is a matter of debate in how far OECs resemble Schwann cells and whether they possess specific properties. Although OECs have been characterized mainly with respect to their regenerative effects after transplantation, both their cellular identity and the regulating factors involved have remained vague. The aim of this article is to define OEC and Schwann-cell identity in molecular terms, and to discuss crucial factors that are involved in determination in vitro and in vivo. Distinct OEC features such as the down-regulation of the low affinity neurotrophin receptor p75(NTR) by neuronal contact are apparent in vivo under physiological conditions, whereas OECs acquire a Schwann cell-like phenotype and up-regulate p75(NTR) expression in vitro and following transplantation into the lesioned spinal cord. This might indicate that establishment of the OEC phenotype depends on specific axonal stimuli. In this review we hypothesize that OECs and Schwann cells possess malleable cellular phenotypes that acquire distinct features only upon specific interaction with their natural neuronal partner.This concept is consistent with previous findings in vitro and in vivo, and might be relevant for studies that use OECs and Schwann cells for nervous system repair.     

Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.     

Morphological phenotypes of olfactory ensheathing cells display different migratory responses upon Slit-2

Olfactory ensheathing cells (OECs) are a type of glial cells with morphological plasticity in the olfactory system. Cultured OECs display the process-bearing and flattened shape. Our previous studies have shown that the frontal application of Slit-2 gradient induced the collapse of leading front, and reversed the soma translocation of process-bearing OECs. However, the migratory properties of flattened OECs upon Slit-2 gradient remain elusive. Here, we found that Slit-2 gradient induced the collapse of their plasma membrane, and inhibited migration of flattened OECs. Upon to Slit-2 gradient, the leading front of flattened type 1 OECs firstly showed collapse and retraction, then gradually re-grew a new lamellipodia, finally, showed collapse again (this phenomenon was called as adaptation), while flattened type 2 OECs only showed collapse of plasma membrane. These different migratory responses upon Slit-2 stimulation were possibly due to their different sub-cellular distribution of Robo receptor. Furthermore, F-actin at the peripheral region of leading front was more sensitive to the Slit-2 stimulation than microtubules and the loss of F-actin might be implicated in initiating the collapse of flattened OECs. Finally, the adaptation of flattened type 1 OECs induced by Slit-2 was independent on protein synthesis. Taken together, these results demonstrate that morphological phenotypes of OECs display different migratory properties upon Slit-2 and an unexpected finding that the protein synthesis-independent adaptation in OECs induced by Slit-2.     

Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord

Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.     

Chronic TNFα Exposure Induces Robust Proliferation of Olfactory Ensheathing Cells,but not Schwann Cells

TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5–7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.     

Cell surface expression of 27C7 by neonatal rat olfactory ensheathing cells in situ and in vitro is independent of axonal contact

Olfactory ensheathing cells (OECs) are Schwann cell-like glial cells of the olfactory system that promote neural regeneration after transplantation into the injured central nervous system. Compared to the closely related Schwann cells, however, the biological characterization of OECs has remained fragmentary. This is due to the fact that the expression of OEC-specific markers is subject to complex regulation and that intricate ultrastructural analysis is essential to determine their localization. The p75 neurotrophin receptor (p75^NTR) as the prototype OEC marker, for example, is only expressed by a minor population of neonatal rat OECs in situ. The major population carries O4-positive axonal fragments on their surface after dissociation and up-regulates p75^NTR during culturing (Wewetzer et al. in Glia 49:577–587, 2005). In the present study, we investigated whether the cell surface determinant 27C7, defined by a monoclonal antibody to Schwann cells, is also expressed by neonatal rat OECs in situ and in vitro. Primary cell suspensions of the olfactory bulb displayed 27C7 expression of both p75^NTR-negative and p75^NTR-positive OECs, while immature oligodendrocytes and astrocytes were devoid of any 27C7 labeling. This together with the finding that the intrafascicular OECs of the olfactory nerves in the mucosa expressed 27C7 but not p75^NTR, suggests that 27C7 was expressed by the entire OEC population in situ. Maintenance of OECs in the absence of olfactory neurons in organotypic slice culture up-regulated p75^NTR but did not alter 27C7 expression. It is concluded that 27C7 unlike p75^NTR is constitutively expressed by OECs and may, therefore, be a useful marker for characterization of neonatal OECs in situ and in vitro.     

Defining the morphological phenotype: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a novel marker for in situ detection of canine but not rat olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are the non-myelinating glial cells of the olfactory nerves and bulb. The fragmentary characterization of OECs in situ during normal development may be due to their small size requiring intricate ultrastructural analysis and to the fact that available markers for in situ detection are either expressed only by OEC subpopulations or lost during development. In the present study, we searched for markers with stable expression in OECs and investigated the spatiotemporal distribution of CNPase, an early oligodendrocyte/Schwann cell marker, in comparison with the prototype marker p75^NTR. Anti-CNPase antibodies labeled canine but not rat OECs in situ, while Schwann cells and oligodendrocytes were positive in both species. CNPase immunoreactivity in the dog was confined to all OECs throughout the postnatal development and associated with the entire cell body, including its finest processes, while p75^NTR was mainly detected in perineural cells and only in some neonatal OECs. Adult olfactory bulb slices displayed CNPase expression after 4 and 10 days, while p75^NTR was detectable only after 10 days in vitro. Finally, treatment of purified adult canine OECs with fibroblast growth factor-2 significantly reduced CNPase expression at the protein and mRNA level. Taken together, we conclude that CNPase but not p75^NTR is a stable marker suitable for in situ visualization of OECs that will facilitate their light-microscopic characterization and challenge our general view of OEC marker expression in situ. The fact that canine but not rat OECs expressed CNPase supports the idea that glia from large animals differs substantially from rodents.     

Schwann-like cells can be induction from human nestin-positive amniotic fluid mesenchymal stem cells

We examined the morphological, phenotypic, and functional characteristics of human amniotic fluid mesenchymal stem cells (AF-MSCs) differentiated towards a Schwann cell lineage. Initially, we induced human AF-MSCs into nestin-positive AF-MSCs. And then, these nestin-positive AF-MSCs were induced into floating neurospheres. After that, neurospheres were induced to differentiate into Schwann-like cells using glia growth factors. In comparison with AF-MSCs, nestin-positive AF-MSCs significantly increased the ratio of neurosphere formation and the percentage of nestin expression in the neurosphere. Differentiated AF-MSCs showed morphological changes similar to those found in Schwann cells. Expression of the Schwann cell markers was determined by immunocytochemical staining and western blotting. Furthermore, differentiated AF-MSCs could promote neurite outgrowth in co-culture with dorsal root ganglia neurons. These results suggest that conversion of human nestin-positive AF-MSCs into cells with Schwann-like cell characteristics is possible and that these cells may have the potential for future cellular therapy for peripheral neurological disorders.