神经干细胞在创伤性脑损伤灶周边的成活和迁移*

背景：成年人中枢神经系统再生困难,颅脑损伤后,损伤灶周边区域神经细胞的存活数量直接影响患者的预后。如何有效地使移植入创伤性脑损伤灶周边的神经干细胞存活分化,是目前神经修复再生研究的重点。 目的：探讨神经干细胞移植入大鼠创伤性脑损伤灶周边的成活、迁移和分化情况。 方法：利用无血清培养技术,加入表皮生长因子、碱性成纤维生长因子诱导刺激大鼠胚胎源性前脑神经干细胞生长增殖,并在体外进行克隆培养,移植前行BrdU标记,采用免疫组化和免疫荧光法检测其增殖特性和多向分化潜能,并观察其移植到Fenney’s落体脑损伤模型鼠脑皮质内的成活和迁移情况。 结果与结论：免疫组化及免疫荧光检测结果显示克隆细胞球呈nestin和BrdU阳性,分化后呈NSE,GFAP,MAP-2阳性。免疫组化及荧光双标检测结果显示移植后7,14 d损伤灶周边散在BrdU阳性细胞,并且GFAP阳性细胞增多。提示前脑神经干细胞在体外培养中能够增殖,并分化为神经元和神经胶质细胞,移植后能够在创伤性脑损伤灶周边存活和迁移,形态上显示出与脑组织整合的特点。     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

食蟹猴脑室下区成体神经前体细胞培养及鉴定

目的分离、培养成年食蟹猴脑室下区神经前体细胞并研究其生物学特性。方法取成年食蟹猴脑室下区组织,经木瓜蛋白酶和脱氧核糖核酸酶消化后,接种于DMEM-F12无血清培养基中。结果神经前体细胞可以增殖形成神经球,经免疫细胞化学方法检验这些细胞呈现巢蛋白nestin阳性,神经球分化后的细胞表达胶质细胞的标记物及神经元的标记物。脑室下区来源的神经球进行自主分化时可分化成神经元和胶质细胞,但分化成神经元的比例都很少。结论从食蟹猴脑室下区分离培养的细胞可以在体外增殖形成神经球,并可分化为神经元和胶质细胞,符合神经前体细胞的生物学性状。     

C57BL／6-gfp小鼠嗅球NSC培养及定位注射方法的建立

目的建立一套稳定的绿色荧光蛋白转基因小鼠（C57BL／6-gfp）嗅球神经干细胞（NSC）体外培养的方法,并初步应用于定位注射试验,为干细胞移植治疗神经性耳聋的实验研究奠定基础。方法培养C57BL／6-gfp小鼠胚胎嗅球NSC,传代并进行分化实验,鉴定后将其立体定位注射于大鼠耳蜗核。结果所培养的NSC生长良好,可稳定传代,能够分化为三种神经细胞。定位注射后可在局部见到绿色荧光阳性的细胞团。结论该方法培养的C57BL／6-gfp小鼠胚胎嗅球NSC可稳定传代并可作为荧光标记细胞进行移植试验。     

大鼠胚胎神经干细胞的体外培养及鉴定

目的探讨大鼠胚胎神经干细胞（NSC）的体外培养和诱导分化的条件和特点。方法从孕14～16d的大鼠胚胎脑皮质中分离NSC，在表皮生长因子、碱性成纤维生长因子和B27联合作用下使其稳定增殖，并用10％的胎牛血清诱导其贴壁分化，应用免疫荧光染色方法行巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）、Gale-C免疫荧光染色，对NSC及其分化的细胞进行鉴定。结果体外培养的NSC增殖成神经干细胞球并传代，鉴定为Nestin染色阳性细胞，并可诱导分化为神经元细胞（NSE染色阳性细胞）、星形胶质细胞（GFAP染色阳性细胞）和少突胶质细胞（Gale-C染色阳性细胞）。结论采用无血清培养基中加入特定生长因子的培养技术，可培养出在体外稳定增殖并有多向分化潜能的大鼠胚胎NSC。     

1101/神经干细胞超顺磁性氧化铁标记及对体外诱导分化的影响

目的 研究超顺磁性氧化铁（Superparam agnetic iron oxide）体外标记人神经干细胞的效果,为将来临床上应用磁共振成像（MRI）追踪标记细胞奠定基础。 方法 取孕13周人胚胎脑组织,制备成单细胞悬液,用添加表皮生长因子（EGF,20ng/ml）和碱性成纤维细胞生长因子（bFGF,20ng/ml）的体外培养人神经干细胞球,并做传代及诱导分化。利用Feridex标记分散成单细胞的神经干细胞,行普鲁士蓝染色检测标记阳性率,5%胎牛血清（FBS）诱导标记后的神经干细胞分化,并用免疫荧光染色检测分化细胞。 结果 培养一周即可见大量的神经干细胞球,Feridex标记神经干细胞行铁染色显示胞浆中含有铁颗粒,Feridex标记的神经干细胞阳性率达90%。标记后的神经干细胞经5% FBS诱导分化能够分化为神经元和星形胶质细胞 。 结论 Feridex 能够标记神经干细胞,标记不影响神经干细胞的生物学特性,标记后能够正常体外扩增培养和定向诱导分化,本研究为神经干细胞临床移植追踪提供了新的方法。     

神经干细胞移植治疗脊髓损伤

目的:探讨神经干细胞移植对脊髓损伤大鼠后肢运动功能修复的影响。方法:SD大鼠36只,制成T10脊髓全横断损伤模型。于造模成功后1周采用局部微量注射法移植。随机分三组：A损伤对照组（n=12）仅打开椎管暴露脊髓;B移植对照组（n=12）：注射10μl DMEM/F12培养液;C细胞移植组（n=12）：移植1.0?06/ml的神经干细胞悬液10μl。移植后通过不同时间点BBB行为评分、病理组织学、免疫荧光技术评价大鼠大鼠脊髓功能修复情况及移植细胞在体内的存活、迁移、分化。 结果:在体外成功建立SD大鼠海马源性神经干细胞培养体系;B、C两组大鼠随着时间延长BBB评分均不同程度提高,从移植后2W起C组大鼠评分明显高于B组,两组比较差异有统计学意义（P<0.05）;神经干细胞移植后能够在体内继续存活、迁移并且分化为NF-200、GFAP表达阳性的神经元及星形胶质细胞。 结论:神经干细胞移植治疗脊髓损伤是一种有效的方法。     

大鼠胚胎神经干细胞移植治疗脑出血的实验研究

目的 研究大鼠胚胎神经干细胞移植治疗脑出血的可行性。方法 从孕龄16天的大鼠胚胎脑组织中分离、培养神经干细胞并诱导其分化，通过免疫组化学技术研究其特性。制作大鼠脑出血模型，3天后将未分化的神经干细胞注入血肿同侧或对侧的尾状核内，记录损伤和移植后的大鼠运动功能。不同时间杀死大鼠，研究移植后的干细胞在体内分化和迁徙的情况。结果 实验中分离、培养的神经干细胞体外能够被诱导分化成神经元、光突胶质细胞和星形胶质细胞，血肿同侧移植干细胞的大鼠运动功能的改善显著好于血肿对侧移植干细胞组及未移植干细胞的对照组。免疫组化方法证实移植后的干细胞在体内可分化成神经元和胶质细胞，并向损伤区域迁徙。结论 大鼠胚胎神经干细胞体内、体外均具有多向分化潜能，其分化成各种类型神经细胞的比例与所处的外界环境有关，在脑内靠近损伤部位移植胚胎神经干细胞后能够有效改善脑出血动物的运动功能。     

神经干细胞脑室内移植治疗大鼠脑缺血的实验研究

目的 探讨大鼠胚胎神经干细胞移植治疗局灶性脑缺血再灌注损伤的可行性。方法 取胎龄8～10d大鼠神经干细胞体外扩增．采用免疫组织化学方法检测神经干细胞及其分化后代的特异性标志巢蛋白（nestin）、胶质纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）的表达。以Brdu标记神经干细胞，分别于缺血后24h和4周移植至局灶性脑缺血大鼠模型的脑室内和梗死中心，比较不同移植部位和不同移植时间神经干细胞的存活、增殖和迁移情况。结果 移植后可见移植细胞存活至少8周以上．移植部位不同不影响细胞存活。脑室内移植细胞向脑缺血区域迁移．且以缺血后24h移植组较缺血后4周移植组迁移趋向性更强。结论 大鼠胚胎神经干细胞移植至局灶性脑缺血大鼠脑室内和梗死中心均可长期存活并广泛迁移．其迁移趋化能力与缺血后移植时间有关。     

大鼠嗅鞘细胞和星形胶质细胞对神经干细胞分化作用的比较研究

目的比较大鼠嗅鞘细胞与星形胶质细胞对神经干细胞分化的影响。方法分别从新生SD大鼠嗅球、海马、皮质分离、培养嗅鞘细胞、神经干细胞和星形胶质细胞。收集嗅鞘细胞、星形胶质细胞及其上清液,分别对神经干细胞(neural stem cells,NSCs)进行诱导分化,倒置显微镜下观察细胞的生长情况,并采用免疫组化法对分化细胞鉴定和计数。结果嗅鞘细胞组分化为神经元比率高于星形胶质细胞组(P<0.01)。结论嗅鞘细胞较星形胶质细胞能更好地促进神经干细胞分化为神经元。     

Adult-derived neural precursors transplanted into multiple regions in the adult brain

Neural stem cells persist in the adult brain subventricular zone (SVZ). These cells generate a large number of new neurons that migrate to the olfactory bulb, where they complete their differentiation. Here, we transplanted cells carrying beta-galactosidase under the control of neuron-specific enolase promoter (NSE::LacZ) from the SVZ of adult mice into the striatum cortex and olfactory bulb, with or without an excitotoxin lesion. Between 2 and 8 weeks after transplantation, grafted cells were present in the recipient regions, but extensive migration and differentiation into mature neurons of grafted cells were only observed in the olfactory bulb. Clusters of graft-derived neuroblasts forming chain-like structures were observed within or close to the grated sites in the cortex and striatum; electron microscopy confirmed that graft-derived cells in the olfactory bulb and a small number in the striatum were neurons. Surprisingly, most of the cells expressing NSE::LacZ outside the olfactory bulb were astrocytes. We conclude that primary precursors from the SVZ migrate and differentiate effectively only within the environment of the olfactory bulb. Only limited survival and differentiation were observed in other brain regions studied.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

Absence of hematopoiesis from transplanted olfactory bulb neural stem cells

Neural stem cells giving rise to neurons and glia cells have been isolated from the embryonic and adult central nervous system. The extent to which they are able to differentiate into cells of non-neural lineages, such as the hematopoietic lineage, is nonetheless unclear. We previously reported the isolation of stem cells from the mouse olfactory bulb neuroepithelium. In the present study, we analysed whether olfactory bulb stem cells (OBSC) can generate cells with hematopoietic features. Cells were prepared from the olfactory bulbs of transgenic mice expressing enhanced green fluorescent protein (EGFP). In culture, transgenic cells proliferated with the same kinetics as wild-type cells. Following mitogen removal, both cell types gave rise to similar numbers of neurons, astrocytes and oligodendrocytes, indicating that EGFP overexpression does not alter OBSC proliferation and differentiation patterns. When these cells were injected into the tail vein of irradiated mice, no hematopoietic cells derived from the OBSC could be recovered in their peripheral blood, spleen or bone marrow. By contrast, when OBSC were transplanted into the adult brain, EGFP-positive cells were found in the striatum and corpus callosum; differentiated cells expressed antigenic markers of neurons and astrocytes. These results suggest that embryonic olfactory bulb stem cells are not endowed with the potential to produce hematopoiesis.     

Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb

In the adult rat olfactory bulb, neurons are continually generated from progenitors that reside in the lateral ventricle wall. This study investigates long-term survival and cell death of newly generated cells within the adult olfactory bulb. After injecting rats at 2 months of age with 5-bromodeoxyuridine (BrdU), the newly generated cells were quantified over a period of 19 months. A peak of BrdU-positive cells was reached in the olfactory bulb 1 month after BrdU injection, when all new cells have finished migrating from the ventricle wall. Thereafter, a reduction of BrdU-positive cells to about 50% was observed and it was confirmed by dUTP-nick end-labelling (TUNEL) that progenitors and young neurons undergo programmed cell death. However, cells that survived the first 3 months after BrdU injection persisted for up to 19 months. The majority of the BrdU-positive cells that reach the olfactory bulb differentiate into granule cells, but a small fraction migrate further into the glomerular layer. These newborn cells differentiate more slowly into periglomerular interneurons, with a delay of more than 1 month when compared to the granule cells. The newly generated periglomerular neurons, among them a significant fraction of dopaminergic cells, showed a similar decline in number compared to the granule cell layer and long-term survival for the remaining new neurons of up to 19 months. Rather than replacing old neurons, this data suggests that adult olfactory bulb neurogenesis utilizes the overproduction and turnover of young neurons, which is reminiscent of the cellular dynamics observed during brain development.     

Host primary olfactory axons make synaptic contacts in a transplanted olfactory bulb

Previous light microscopic studies have shown that host olfactory neurons are able to grow into a transplanted fetal olfactory bulb, and behavioral studies have shown that animals with transplanted olfactory bulbs recover functional olfactory abilities. We examined the olfactory bulb transplant at the ultrastructural level to determine whether synaptic contacts are reestablished between host olfactory neurons and donor olfactory bulb. Mature rats that, as neonates, had received embryonic olfactory bulb transplants following olfactory bulb removal were studied. An antibody specific for olfactory marker protein was used to identify the primary olfactory neurons; it was bound by a gold-conjugated secondary antibody for visualization. To preserve the antigenicity of the olfactory marker protein for immunolabeling, Lowicryl K4M hydrophilic resin was used. Synaptic contacts were unmistakable between labeled axons of host olfactory neurons and unlabeled processes within glomerulus-like areas of the transplanted olfactory bulb. The surrounding neuropil contained other elements similar to those found in normal tissue, including synaptic contacts between unlabeled profiles. We clearly show that the transplanted olfactory bulb exhibits sufficient plasticity to form an array of normal synaptic contacts, including the contacts from host primary olfactory neurons. © 1995 Wiley-Liss, Inc.     

胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋白损伤大鼠海马后的靶向迁移与分化

目的观察小鼠胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋（amyloidβpeptide,Aβ）损伤大鼠海马后的靶向迁移及在体分化。方法采用无血清培养法将表达绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的小鼠胚胎干细胞定向诱导为神经前体细胞,移植至Aβ1-40单侧损伤的大鼠海马;免疫荧光观察移植细胞的迁移距离、迁移方向与分化情况;对移植细胞的平均迁移距离与平均分化率作相关性分析。结果胚胎干细胞经改良的无血清培养法生长可分化为nestin阳性神经前体细胞。移植后第16周,神经前体细胞平均迁移距离比第4周增长了约5倍。移植细胞中,胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）阳性细胞的平均分化率从（30．41±1．45）％增长到（49．25±1．23）％;神经丝蛋白200（neurofilament200,NF200）细胞的平均分化率从（16．68±0．95）％增长到（27．94±1．21）％;靶向迁移至Aβ斑块同侧的GFAP阳性细胞的比例从60．2％增长到81．3％,靶向迁移至Aβ斑块的NF200阳性细胞的比例从61．3％增至84．1％。相关性分析结果显示平均迁移距离与神经元分化率之间存在显著线性相关（r=0．991）,与胶质细胞分化率之间也存在显著线性相关（r=0．953）。结论胚胎干细胞来源的神经前体细胞移植Aβ损伤模型大鼠海马后能够靶向迁移至Aβ损伤区并分化为胶质细胞和神经元。     

Putative glutamatergic and/or aspartatergic cells in the main and accessory olfactory bulbs of the rat

The "transmitter-specific" retrograde axonal tracer 3H-D-aspartate has been used to demonstrate neurons in the olfactory bulb which putatively utilize aspartate and/or glutamate as their neurotransmitter and which send an axon either to the piriform cortex or within the bulb itself. Injections of 3H-D-aspartate into layer I of the anterior piriform cortex, in the zone of termination of axons from the olfactory bulb, labeled only a few cells in the main olfactory bulb, located in the mitral and external plexiform layers. Although these cells resembled mitral and tufted cells, they tended to have smaller somata than other mitral or tufted cells and apparently form a distinct subpopulation of relay cells. In contrast, many of the mitral cells of the accessory olfactory bulb were labeled by the same injections of 3H-D-aspartate, probably as a result of involvement of the accessory olfactory tract or its bed nucleus in the injection site. Similar injections of the "nonspecific" tracer HRP into the anterior piriform cortex labeled most of the cells in the mitral cell layer of both the main and accessory olfactory bulbs, and some tufted cells in the external plexiform layer. It is concluded that only a small, distinct subpopulation of the mitral or tufted cells of the main olfactory bulb are aspartatergic and/or glutamatergic, while many (at least) of the mitral cells of the accessory olfactory bulb use the excitatory amino acids as transmitters. Injections of 3H-D-aspartate directly into the main olfactory bulb also failed to label the mitral and deeply situated tufted cells. However, a few cells were labeled in the periglomerular region, the superficial external plexiform layer, and the granule cell layer near the injection site. These labeled cells were smaller than mitral and tufted cells but generally larger than periglomerular or granule cells. They may represent a population of glutamatergic or aspartatergic short axon cells. In addition, small cells of an unknown type were labeled in the olfactory nerve layer following injections in the deepest part of the bulb. These cells do not correspond to any of the well characterized cell types of the olfactory bulb.     

Transfer of the von Hippel-Lindau gene to neuronal progenitor cells in treatment for Parkinson's disease

Neuronal progenitor cells (NPCs) may provide dopaminergic neurons for the treatment of Parkinson's disease (PD). However, transplantation of NPCs into the striatum by current methods has had limited success. It is possible to reverse the symptoms of PD in model rats but difficult to reverse them in humans because the number of dopaminergic neurons generated from NPCs is low. We transduced the von Hippel-Lindau (VHL) gene into NPCs isolated from embryonic rat brain. The NPCs with the transduced VHL gene efficiently differentiated into tyrosine hydroxylase-positive neurons in vitro. NPCs with the transduced VHL gene, which were labeled in advance with bromodeoxyuridine, were transplanted into the striatum of a rat model of PD. Numerous bromodeoxyuridine-tyrosine hydroxylase double-labeled cells were seen close to the transplant site, showing that the transplanted cells efficiently generated new dopaminergic neurons within the host striatum. Moreover, all of the animals with NPCs with VHL showed a remarkable decrease in apomorphine-induced rotations. These findings show that NPCs with the VHL gene can efficiently generate dopaminergic neurons and that a sufficient number of dopaminergic neurons can develop from them to reverse the symptoms of PD in humans. VHL gene transduction provides a new therapeutic approach for treatment of PD.     

Differentiation and survival of rat olfactory epithelial neurons in dissociated cell culture

Olfactory epithelial neurons in mammals are unique in that they continue to differentiate from precursor cells in the adult. These neurons extend long axons into the olfactory bulb. Previous attempts to grow these cells in dissociated cell cultures at low density, have not been entirely satisfactory. We report that when plated at very low density, rat olfactory epithelial neurons will differentiate morphologically and biochemically when cultured on astrocytes, but not on non-cellular substrata, such as polylysine, laminin or fibronectin. We demonstrate with antibodies, that these olfactory epithelial neurons require N-CAM, N-cadherin and L1 for neurite extension. Furthermore, synthetic cadherin-peptides containing the tripeptide HAV which is found in the first extracellular domain of N-cadherin, as well as the amino acids flanking this region, appear to be important in cadherin-mediated neurite growth on astrocytes. Astrocytes also appear to enhance the survival and differentiation of olfactory epithelial neurons from embryonic day 15 and 4-5 week post-natal rats, but this effect is not sustained beyond 5 days in cultures of postnatal epithelium.