混合培养对O2A祖细胞定向分化的影响

目的:探讨少突胶质前体细胞在体外采用混合与纯化培养条件向成熟阶段定向分化过程中的形态学差异。方法:新生SD大鼠取大脑皮质原代培养获得混合胶质细胞,以B104神经胶质瘤细胞株条件培养液增殖并获取纯化的少突胶质前体细胞,然后混合胶质细胞组和纯化少突胶质细胞组同时更换无血清的化学条件培养基,倒置相差显微镜观察记录每天细胞的形态变化,分化成熟的少突胶质细胞作扫描电镜观察,并进行免疫细胞化学测定。结果:纯化的少突胶质前体细胞与和星形胶质细胞混合培养者比较,定向分化后细胞突起的长度长、级数多。结论:混合培养中星形胶质细胞的存在可能延缓了少突胶质细胞向成熟阶段的分化。     

新生SD大鼠少突胶质细胞前体细胞的培养与分化

目的 体外培养高纯度的少突胶质前体细胞（oligodendrocyte progenitor cells,OPCs）以及分化成熟的少突胶质细胞（Oligodendrocytes,OLs）.方法新生1 -2 d Sprague-Dawley（SD）大鼠的大脑皮层胶质细胞混合培养9d后采用恒温摇床振荡分离与差异贴壁方法并结合条件培养基纯化培养细胞.光学显微镜观察细胞形态;纯化培养3 d后免疫荧光染色鉴定细胞类型.结果 获得高纯度的少突胶质前体细胞以及成熟的少突胶质细胞,少突胶质前体细胞免疫荧光NG2＋A2B5双标阳性,成熟少突胶质细胞免疫荧光MBP染色阳性.结论 采用恒温摇床振荡分离与差异贴壁法并结合条件培养基可以得到高纯度的少突胶质前体细胞以及成熟少突胶质细胞.     

新生大鼠大脑少突胶质细胞系的分离纯化和定向分化培养

在体外培养条件下获取纯度较高的少突胶质细胞系细胞并探索其分化规律。新生大鼠大脑皮质原代混合胶质细胞培养７－９d或１４－１５d,利用振荡和差速贴壁分离纯化少突胶质前体细胞,再进行无血清化学条件培养基培养。原代培养７－９d时细胞分层形成,Ｏ－２Ａ组细胞（oligodendrocyte/type 2 astrocyte progenitor)分散位于星形质细胞单层上,振荡分离后纯化率大于９５％,分离后的Ｏ－２Ａ祖细胞神经节苷脂ＧＤ３抗体阳性,无血清培养３d后分化为“蜘蛛状”细胞,半乳糖脑脂抗体阳性。原代培养１４－１５d时,星形胶质细胞层上出现葡萄样前Ｏ－２Ａ祖细胞和少量师星形胶质细胞培养时细胞完全分层的出现是振荡分离Ｏ－２Ａ祖细胞的适宜条件,分离纯化的Ｏ－２Ａ祖细胞可自动分化为少突胶质细胞。     

大鼠脊髓少突胶质前体细胞的培养与鉴定方法

目的 探讨大鼠脊髓少突胶质前体细胞（OPCs）的分离纯化及诱导分化方法.方法 取出生后3d内SD大鼠脊髓,采用0.125％胰蛋白酶消化获取原代混合胶质细胞,培养10 d左右在37℃恒温摇床采取180 r/min摇速振摇并差速贴壁40 min获得纯化的OPCs;取纯化后培养3d的OPCs免疫荧光鉴定细胞纯度或诱导分化.结果 采用胰蛋白酶消化法获取原代混合胶质细胞、振摇并差速贴壁法分离纯化能够得到纯度较高的OPCs,折光性强,呈双极或三极形态,免疫荧光染色显示95％细胞表达A2B5和NG2（OPCs标志物）,经诱导分化后表达O4及MBP（少突胶质细胞标志物）.结论 采用振摇差速贴壁法可从大鼠脊髓中分离纯化获得高纯度的OPCs,获得的OPCs体外生长稳定,经诱导可分化为成熟的少突胶质细胞.     

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。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

GFP^＋-大鼠胚胎脊髓源神经干细胞的培养、分化和Neuregulin-1的表达

目的:从GFP -大鼠胚胎脊髓分离和培养神经干细胞(NSC),观察NSC的分化功能和Neuregulin-1的表达。方法:从孕16d的GFP -大鼠胚胎脊髓中分离、培养神经干细胞,用免疫组织化学方法观察神经球的Neuregulin-1表达及鉴定分化的细胞类型。结果:从大鼠胚胎脊髓能分离、培养出NSC。神经球能表达Neuregin-1和Nestin,并能进一步分化为神经元、星形胶质细胞和少突胶质细胞。结论:从GFP -大鼠胚胎脊髓能分离和培养出NSC,该NSC具有分化为用于治疗中枢神经疾病的多种神经细胞的潜能。     

EGb761对慢性脑缺血大鼠脑组织神经胶质细胞的影响

目的 探讨EGb761(银杏叶提取物)对慢性脑缺血大鼠脑组织神经胶质细胞的影响.方法 将雄性SD大鼠随机分为假手术组、单纯缺血组、EGb761干预组.制备慢性脑缺血大鼠模型,药物干预12周后,免疫组化方法分别检测各组大鼠脑组织中星形胶质细胞和少突胶质细胞的表达.结果 EGb761干预组大鼠的海马、胼胝体、皮质CNPase阳性细胞表达明显高于单纯缺血组,而海马、胼胝体、皮质GFAP阳性细胞表达明显低于单纯缺血组(P<0.05),差异有统计学意义.结论 慢性脑缺血时,EGb761对少突胶质细胞缺血性反应有保护作用,同时减少反应性星形胶质细胞增生.     

血管性认知障碍小鼠脑内少突胶质细胞再生分化障碍特点研究

目的 观察血管性认知障碍小鼠模型中,缺血性炎性损伤对室管膜下区及海马齿状回少突胶质细胞 再生分化的影响,为血管性认知障碍的缺血性炎症机制提出新的损伤途径。 方法 成年雄性CD1小鼠随机分为模型组和假手术组,每组24只,模型组采用双侧颈动脉反复缺 血再灌注法制备血管性认知障碍小鼠模型。造模后4～6 d连续腹腔注射5 -溴脱氧尿嘧啶核苷 （bromodeoxyuridine,BrdU）（150 mg/kg）标记新生细胞,分别于术后14 d和28 d每组随机取一半小鼠脑 组织进行脑切片免疫组化、免疫荧光双标共聚焦检测,标记脑组织室管膜下区和海马区的少突胶质 细胞、星形胶质细胞及神经元,观察新生少突胶质细胞增殖及分化情况,并观察星形胶质细胞的增 生活化情况。 结果 造模后14 d和28 d室管膜下区新生细胞（BrdU阳性细胞）在模型组较假手术组明显增加（P均 ＜0.001),造模28 d模型组新生神经元（BrdU/NeuN阳性细胞）较假手术组显著增加（P＜0.001)。与假 手术组相比较,术后28 d模型组海马齿状回少突胶质细胞祖细胞显著增多（P＜0.001）;少突胶质细 胞前体细胞显著减少（P =0.006）。造模后28 d模型组海马齿状回新生星形胶质细胞（BrdU/GFAP阳性 细胞）较假手术组显著增加（P =0.015）。 结论 血管性认知障碍小鼠内源性新生细胞增殖区室管膜下区与海马齿状回区均存在新生细胞反 应性增生的情况。新生细胞区分化的主要细胞为星形胶质细胞,而少突胶质细胞分化障碍,可能是血 管性认知障碍患者影像学常见皮层下白质病变的重要原因。     

SD大鼠星形胶质细胞的原代培养

目的 设计原代培养SD大鼠星形胶质细胞。方法 取新生第2-3dSD红皮鼠，无菌操作下取大脑皮质，剪碎、胰酶消化结合机械吹打使细胞分散，网筛过滤，差速粘附处理去除成纤维细胞，将未粘附的细胞悬液接种培养9～12d，置摇床．舍弃含脱落细胞的细胞悬液，以去除少突胶质细胞和小胶质细胞。细胞传代，GFAP免疫组化染色鉴定。结果 功分离培养了原代神经胶质细胞，并进行了星形胶质细胞的纯化，GFAP鉴定星形胶质细胞比例为95％以上。结论 建立星形胶质细胞体外培养方法，总结操作关键，以此为实验材料对于比较学研究是非常有帮助的。     

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from adult rat spinal cord: In vitro characteristics and response to PDGF,bFGF and NT-3

We have analysed in detail the properties of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from the spinal cords of adult rats to gain further insights into the mechanisms that control the generation of oligodendrocytes in the healthy and demyelinated adult central nervous system (CNS). When O-2A progenitor cells from adult spinal cord are exposed in vitro to the AA homodimeric form of platelet-derived growth factor (PDGF-AA), they express a unipolar morphology, an O4-positive, vimentin-negative antigenic phenotype, divide at slow rates, and appear to generate oligodendrocytes by asymmetric division and differentiation. Furthermore, exposure of these cells to PDGF-AA is sufficient to stimulate their proliferation at clonal density. When adult spinal cord O-2A progenitor cells are exposed simultaneously to PDGF-AA and basic fibroblast growth factor (PDGF/bFGF), they are almost completely inhibited from differentiating into oligodendrocytes, divide more rapidly than cells treated with PDGF-AA, and express a bipolar morphology and an O4-negative, vimentin-positive antigenic phenotype. These findings indicate that adult spinal cord O-2A progenitor cells resemble in many aspects their well-characterised adult optic nerve counterparts. In addition, evidence is presented to indicate that neurotrophin-3 (NT-3) is not mitogenic for adult spinal cord O-2A progenitor cells and that it does not enhance their proliferative response to PDGF-AA or PDGF/bFGF. Since relatively large numbers of O-2A progenitor cells can be obtained from adult spinal cord, it should facilitate the further characterisation of these cells. © 1996 Wiley-Liss, Inc.     

GD3+ cells in the adult rat optic nerve are ramified microglia rather than 0–2Aadult progenitor cells

The adult central nervous system (CNS) contains a population of adult oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells (O-2A^adult progenitor cells). These cells may provide a source of the new oligodendrocytes that are needed to repair demyelinated lesions. In order to examine the role of O-2A^adult progenitor cells in the regeneration of the oligodendrocyte population following demyelinating damage, it is essential to be able to identify such cells unambiguously in sections of adult CNS tissue. The present study examined whether antibodies to the ganglioside G_D3 specifically label O-2A^adult progenitor cells in cultures and sections of adult optic nerve, since previous studies on the developing CNS had suggested that O-2A^perinatal progenitor cells were G_D3⁺ in vitro and in vivo. Evidence is presented indicating that, although O-2A^adult progenitor cells in vitro were labelled with the R24 mAb (an anti-G_D3 mAb), all G_D3⁺ cells in sections of adult optic nerve bound the OX-42 mAb and the B₄ isolectin derived from Griffonia Simplicifolia, and thus were not O-2A^adult progenitor cells, but ramified microglia. The data suggest that O-2A^adult progenitor cells become G_D3⁺ when placed in culture and that ramified microglia lose G_D3-expression in vitro. © 1994 Wiley-Liss, Inc.     

Cortical neurogenesis in adult rats after ischemic brain injury: most new neurons fail to mature

The present study examines the hypothesis that endogenous neural progenitor cells isolated from the neocortex of ischemic brain can differentiate into neurons or glial cells and contribute to neural regeneration. We performed middle cerebral artery occlusion to establish a model of cerebral ischemia/reperfusion injury in adult rats. Immunohistochemical staining of the cortex 1, 3, 7, 14 or 28 days after injury revealed that neural progenitor cells double-positive for nestin and sox-2 appeared in the injured cortex 1 and 3 days post-injury, and were also positive for glial fibrillary acidic protein. New neurons were labeled using bromodeoxyuridine and different stages of maturity were identified using doublecortin, microtubule-associated protein 2 and neuronal nuclei antigen immunohistochemistry. Immature new neurons coexpressing doublecortin and bromodeoxyuridine were observed in the cortex at 3 and 7 days post-injury, and semi-mature and mature new neurons double-positive for microtubule-associated protein 2 and bromodeoxyuridine were found at 14 days post-injury. A few mature new neurons coexpressing neuronal nuclei antigen and bromodeoxyuridine were observed in the injured cortex 28 days post-injury. Glial fibrillary acidic protein/bromodeoxyuridine double-positive astrocytes were also found in the injured cortex. Our findings suggest that neural progenitor cells are present in the damaged cortex of adult rats with cerebral ischemic brain injury, and that they differentiate into astrocytes and immature neurons, but most neurons fail to reach the mature stage.     

Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions

In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes. In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons. Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse. Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats. Cell suspensions of primary LGE tissue were prepared and grafted in parallel. Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes. Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons. Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb. MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus. The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.     

AP-1 activity during the growth, differentiation, and death of O-2A lineage cells

Oligodendrocyte differentiation has been correlated with AP-1 activity, being low in progenitors and high in differentiated cells. In this study we have carried out a detailed temporal analysis of AP-1 activity in oligodendrocyte-type-2 astrocyte (O-2A) lineage cells. We show that low AP-1 activity in progenitor cells depended on the application of growth factors. Treatment of cells with B104-conditioned medium induced high AP-1 activity, increased process length, and improved growth. The role of AP-1 in proliferation and process extension was emphasized when progenitor cells overexpressing a c-Jun dominant-negative mutant had impaired growth and shortened processes. AP-1 DNA-binding activity during O-2A differentiation in vitro showed an initial down-regulation followed by up-regulation after 2 days. Increased AP-1 levels in oligodendrocytes were inhibited by overexpression of bcl-2, indicating that AP-1 in mature oligodendrocytes is involved in the regulation of apoptosis. Prevention of cell death by bcl-2 in oligodendrocytes was accompanied by progressive differentiation and expression of MOG and PLP.     

大鼠背根神经节细胞的纯化培养

目的:在NB1培养基中建立体外胚胎大鼠背根神经节(DRG)细胞的纯化培养体系。方法:取胎鼠的背根神经节,用胰蛋白酶消化法分离成单细胞,通过差速贴壁法进行背根神经节神经元的分离纯化,在NB1中培养,用活细胞计数和神经元特异性的烯醇化酶(NSE)免疫组织化学染色相结合判定培养神经元的纯度。结果:纯化培养神经元生长状态良好,培养4天时神经元纯度为91％左右。结论:采用差速贴壁法可获得高纯度的神经元,可作为神经科学研究的重要工具。     

In vitro identification and functional characterization of glial precursor cells in human gliomas

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.