新生大鼠大脑皮质O-2A祖细胞的体外诱导分化

目的　在获取高纯度O 2A祖细胞的基础上 ,探讨O 2A祖细胞在体外分化的形态学特点。方法　采用“两次恒温摇床振荡法”和差速贴壁法 ,结合使用神经营养因子 (bFGF、PDGF AA) ,进行O 2A祖细胞体外纯化和扩增培养 ,并观察O 2A祖细胞在有血清和无血清培养条件下的分化情况 ,用免疫细胞化学鉴定分化成熟的 2型星型胶质细胞和少突胶质细胞。结果　培养的O 2A祖细胞能形成克隆球 ,具有增殖能力 ,经免疫细胞化学鉴定细胞纯度达 90 %以上。在含血清的培养基中能定向分化为 2型星形胶质细胞 ,其胞体较大 ,从胞体上伸出细长突起 ,由突起上再发出更为细小的分支 ,GFAP和A2B5抗体标记均为阳性 ;在无血清的化学条件培养基中能定向分化为少突胶质细胞 ,其胞体较小 ,突起短而细 ,相互交织呈“蜘蛛网”样 ,CNPase抗体标记阳性。结论　 (1)培养的O 2A祖细胞保持定向干细胞的特性 ,具有增殖和双向分化的潜能 ;(2 )O 2A祖细胞定向分化为 2型星形胶质细胞过程中经历较长的增殖期 ,而分化为少突胶质细胞过程中则增殖期较短 ;(3)分化的 2型星形胶质细胞和少突胶质细胞在形态学和抗原表达上存在差异。     

体外培养获取高纯度O-2A祖细胞

目的获取较高纯度的O-2A祖细胞,并观察在不同的实验条件下O-2A祖细胞的分化作用。方法 根据星形胶质细胞、O-2A祖细胞的生长时间差异,细胞的粘附特性不同,采用振荡和差速贴壁法结合Ⅰ型星形胶质细胞条件培养液,培养、分离纯化O-2A祖细胞。结果培养的o-2A祖细胞胞体呈椭圆形,有典型的双极突起,少数呈三极突起;经免疫组织化学染色鉴定细胞的纯度达90％。在有血清的情况下,O-2A祖细胞定向分化为Ⅱ型星形胶质细胞;在无血清的情况下,其定向分化为少突胶质细胞。结论通过振荡法和Ⅰ型星形胶质细胞条件培养液结合差速贴壁法分离纯化培养O-2A祖细胞是一种可行的培养方法。     

IGF-1诱导脐血间充质干细胞向少突胶质祖细胞分化的研究

目的 探讨胰岛素样生长因子-1(IGF-1)对脐血间充质干细胞(MSCs)诱导分化的影响.方法 第3代纯化的脐血MSCs在含有或无IGF-1的培养基条件下贴壁培养10d,免疫荧光显色,观察分化为少突胶质祖细胞(02A)的数量.结果 脐血MSCs在不含IGF-1培养基的条件下分化为少突胶质祖细胞的数量较少,而在含有IGF-1的培养基的条件下分化为少突胶质祖细胞的数量明显增加.结论 IGF-1可以诱导脐血MSCs向少突胶质祖细胞分化.     

少突胶质谱系细胞的生物学特性

目的研究少突胶质谱系细胞定向分化过程中的增殖及形态学的变化,为进一步研究脑室周围白质软化的临床治疗奠定基础。方法依据各种胶质细胞黏附性及生长时间的差异,通过机械振荡法和差速贴壁法从原代培养中获得纯化的O-2A祖细胞,接种于含有血小板源性生长因子和碱性成纤维细胞生长因子的培养液观察其增殖情况,再以三碘甲状腺原氨酸和睫状神经营养因子定向诱导分化,并观察分化过程中细胞的形态学变化,免疫细胞化学鉴定。结果纯化的O-2A祖细胞接种后指数式增殖呈团,细胞界限不清,表达NG2,至第4天细胞不再增多进入分裂终期;进入分化过程的少突胶质谱系细胞突起逐渐分级、交互,免疫化学鉴定依次表达O4、Gal C及MBP。结论 O-2A祖细胞具有良好的增殖能力,沿着少突胶质谱系细胞定向分化伴随形态学及表面表达抗原的变化。     

少突胶质细胞体外培养、纯化、鉴定及缺氧模型建立

目的观察体外培养少突胶质细胞的增殖、分化及其生物学特性;探讨获得细胞纯度较高的实验方法及如何建立少突胶质细胞的缺氧培养模型。方法1．取新生SD大鼠脑皮质进行体外培养,采用两次恒温摇床振荡分离纯化法和条件限定培养基培养,倒置显微镜结合免疫荧光染色法鉴定细胞种类并分析纯度。2．缺氧联合低糖培养建立细胞体外缺氧培养模型,相差显微镜观察细胞的形态学变化。结果1．获取高纯度的大鼠少突胶质细胞。少突胶质前体细胞祖细胞A285荧光标记阳性,成熟少突胶质细胞半乳糖脑苷脂阳性。2．缺氧培养2小时细胞变化不明显,但缺氧培养6小时,细胞存活率明显下降,并且随着缺氧时间延长存活细胞更少,凋亡细胞更多,至缺氧培养10小时,大多数细胞破碎。结论1．两次恒温摇床振荡分离纯化法和条件限定培养基培养可获取高纯度的大鼠少突胶质细胞。2．低糖联合缺氧培养模型可以建立可行的少突胶质细胞体外缺氧模型。     

少突胶质细胞发生过程中S-100β表达的变化

目的:探讨少突胶质细胞发生过程中3个主要不同阶段S-100β的表达.方法:采用振荡法和差速贴壁法,获得纯化的O-2A祖细胞;双重免疫荧光细胞化学显色法观察S-100β在少突胶质细胞发生过程中的表达情况.结果:O-2A祖细胞在含有碱性成纤维细胞因子(10 ng/ml)和血小板源性生长因子(10 ng/ml)培养液中培养48 h,不表达S-100β;换成含有3'碘甲状腺原氨酸(30 ng/ml)培养液继续培养24 h,S-100β免疫反应呈现阳性.前少突胶质细胞、少突胶质细胞中表达S-100β.结论:少突胶质细胞发生过程中S-100β的表达始于O-2A祖细胞过渡到前少突胶质细胞之间,可能与其从多潜能分化阶段过渡到形态学上的分化阶段有关.     

一种分离培养少突胶质前体细胞的改进方法

目的:对少突胶质前体细胞的分离培养方法进行改良,为今后细胞移植治疗研究提供基础。方法:在大鼠少突胶质前体细胞分离纯化培养过程中添加不同浓度bFGF(5、10、20 ng/ml),显微镜观察少突胶质前体细胞在体外的生长情况,台盼蓝实验检测细胞存活率和获得率,免疫细胞化学技术进行细胞鉴定,流式细胞术检测细胞纯度。结果:在少突胶质前体细胞培养过程中添加10 ng/ml的bFGF,可明显提高少突胶质前体细胞的产出率和纯度。分离的少突胶质前体细胞呈A2B5、NG2阳性,能进一步分化为成熟少突胶质细胞且表达MBP和O4。结论:在培养中添加适当浓度的bFGF的方法能有效获得高纯度的少突胶质前体细胞。     

少突胶质细胞与2型星形胶质细胞中S-100β表达差异性的研究

目的研究少突胶质细胞(oligodendrocytes,OL)与2型星形胶质细胞(type-2astrocyte,T2A)中S-100的表达。方法根据各种胶质细胞的生长时间的差异及细胞的黏附性不同,通过机械振荡法和差速贴壁法获得纯化的O-2A祖细胞;免疫细胞化学染色法观察S-100β在O-2A谱系细胞中的表达情况。结果O-2A祖细胞在含有PDGF、bFGF的培养液中培养3d,分化成T2A,培养5d分化成OL;OL中S-100β免疫反应呈现阳性,T2A中不表达S-100β。结论O-2A谱系细胞中的S-100β的表达与O-2A祖细胞向OL分化有关。     

蛋白脂蛋白在两型星形胶质细胞和少突胶质细胞中的表达

目的:探讨蛋白脂蛋白(PLP)在分化成熟的两型星形胶质细胞和少突胶质细胞中的表达。方法:用激光共聚焦双重免疫荧光标记技术检测PLP在分化成熟的两型星形胶质细胞和少突胶质细胞中的表达情况。结果:在O-2A谱系来源的成熟2型星形胶质细胞和少突胶质细胞中PLP抗体标记为阳性,而在T1A谱系来源的成熟1型星形胶质细胞中则未检测到PLP的表达,从而在蛋白质水平验证本室研究两型星形胶质细胞基因表达谱差异基因芯片结果中PLP mRNA在T2A中高表达,而在T1A中低表达的现象。结论:PLP等脂代谢相关基因可能在O-2A谱系发生和分化过程中起重要作用,O-2A谱系与神经髓鞘发生以及脑内脂质代谢内在机制密切相关。     

少突胶质祖细胞移植对新生鼠缺氧缺血脑损伤学习记忆功能的影响

目的探讨原代少突胶质祖细胞(O2A)的培养方法,并移植入缺氧缺血新生鼠脑内,为细胞干预早产儿白质软化病的研究提供细胞学基础。方法根据O2A的生长时间、生长方式及细胞对培养层粘附等特性的差异,采用两次恒温摇床振荡分离纯化法和条件限定培养基培养,获得高纯度的O2A,经脑室移植入缺氧缺血性脑白质损伤的新生鼠脑内。结果体外培养24h后,细胞成堆聚集形成克隆球;第2～3天,细胞呈圆形或椭圆形,多数具有双极突起。实验新生鼠由脑室移植O2A后,测定平均逃避潜伏期:PVL+O2A移植组为(7.33±1.41)s,PVL+PBS组为(16.24±1.63)s,PVL组为(11.75±1.50)s;PVL+O2A移植组的平均逃避潜伏期比PVL+PBS组和PVL组缩短,与PVL+PBS组、PVL组比较差异均有统计学意义(P<0.001)。结论两次恒温摇床振荡分离纯化法和条件限定培养基培养可获取高纯度的O2A;平均逃避潜伏期测定提示,移植O2A在脑内可分化为成熟的少突胶质细胞,使轴突重新髓鞘化,促进脑功能的修复。     

B104CM对早期培养时大鼠O2A祖细胞迁移的促进作用

目的：研究B104CM对体外培养条件下的少突胶质前体细胞的促迁移作用。方法：取新生SD大鼠大脑皮质原代混合培养少突胶质前体细胞,B104神经胶质瘤细胞株条件培养液增殖并纯化,琼脂糖滴迁移实验观察B104CM对少突胶质前体细胞迁移的影响。结果：B104CM实验组可以促进少突胶质前体细胞的迁出,迁移速度有时段上的差别。结论：B104CM对体外培养的少突胶质前体细胞有增殖作用的同时,还可促进其迁移。     

Oligodendrocyte progenitor cells derived from mouse embryonic stem cells give rise to type-1 and type-2 astrocytes in vitro

Oligodendrocyte progenitor cells (OPCs) in primary culture can give rise to mature oligodendrocytes and type-2, but not type-1 astrocytes depending on the culture conditions. The OPCs thus are called oligodendrocyte-type-2 astrocyte (O2-A) progenitor cells. Mouse embryonic stem cells (mESCs) have been efficiently differentiated into OPCs; however, the fate plasticity of mESC-derived OPCs is not well characterized. In the present study, using GFP-Olig2 mESC line, we showed that the Olig2(+)/GFP(+)/A2B5(+)/NG2(+) OPCs derived from GFP-Olig2 mESCs can mature into oligodendrocytes when co-cultured with mESC-derived neurons. Interestingly, when induced to astrocytic differentiation by bone morphogenetic protein-4, these mESC-derived OPCs can not only generate type-2 astrocytes, but also type-1 astrocytes. These results challenge the dogma that OPCs in culture can only generate type-2, but not type-1 astrocytes, and support the in vivo finding that during perinatal development, OPCs can give rise to a subset of type-1 astrocytes.     

Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.     

Regulation of Notch signaling during T- and B-cell development by O-fucose glycans

Summary:  Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O -fucose glycans of Notch cell-autonomously modulate Notch–ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O -fucosyltransferase 1 (Pofut1), and elongated by the transfer of N -acetylglucosamine (GlcNAc) to the fucose by β1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O -fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O -fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O -fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.     

IL-6 attenuates apoptosis, while neither IL-6 nor IL-10 affect the numbers or protease phenotype of fetal liver-derived human mast cells

BACKGROUND: The combination of recombinant human stem cell factor (rhSCF), rh interleukin (IL)-6 and rhIL-10 was reported to be optimal for mast cell development from cord blood progenitors and to induce chymase expression in all such mast cells earlier in their development than tryptase. OBJECTIVE: The effects of rhIL-6 and rhIL-10 in various combinations on the rhSCF-dependent development of human mast cells from fetal liver progenitors were examined in serum-free media. METHODS: Dispersed fetal liver cells were cultured in serum-free AIM-V medium with rhSCF alone, or with combinations of rhIL-6 and rhIL-10. Tryptase and chymase expression, surface Kit expression, metachromasia with toluidine blue and apoptosis were measured. RESULTS: Neither rhIL-6 nor rhIL-10 nor the two interleukins together, when included from day 0 of culture, affected the number or protease phenotype of mast cells at 1 or 3 weeks. Expression of tryptase paralleled the appearance of metachromasia and surface Kit, both of which preceded chymase expression, regardless whether a rabbit polyclonal or mouse monoclonal anti-chymase antibody preparation was used. On the other hand, rhIL-6 markedly attenuated baseline levels of apoptosis in the presence of rhSCF as well as apoptosis occurring after withdrawal of rhSCF, whereas rhIL-10 had no effect. CONCLUSION: RhIL-6 protected fetal liver-derived mast cells from apoptosis, particularly after withdrawal of rhSCF, but neither rhIL-6 nor rhIL-10 nor the combination of these interleukins affected the numbers or protease phenotype of these mast cells.     

Studies on Biological Characteristics of Rabbit Oral Mucosal Epithelial Cells

Objective:The biological characteristics of rabbit oral mucosal epithelial cells(OMECs) were studied and the suitable procedure for the culture of OMECs was explored.Methods:Cultured OMECs by different surface disinfection,by different Dispase Ⅱ preparation,in different medium,in different calciumion concentration of K-SFM medium.Results:Microbial contamination of cell culture could significantly reduce with 2% iodine solution used for sterilization of oral cavity before cell culture.The OMECs digested with Dispase Ⅱin K-SFM could attach to the culture flask quickly,the OMECs digested with Dispase Ⅱ in PBS rare attached to the culture flask.The proliferations of OMECs cultured in 0 or 0.09 mmol/L calcium concentrations of K-SFM medium showed not statistically significant.the OMECs grew fast with serum,but it was easy to differentiate into fibroblast-like cells.Conclusion:OMECs could grow well and had the typical oral mucosal epithelial cell morphology by the procedure that described in this experiments.