大鼠嗅球神经干细胞培养

目的建立大鼠嗅球神经干细胞(neural stem cells，NSC)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、培养新生第1天和成年大鼠嗅球NSC，应用免疫细胞化学方法鉴定培养的NSC及自然分化为特异性神经细胞的类型，四甲基偶氮唑盐(MTY)法测定嗅球NSC的生长曲线。结果从生后第1天和成年大鼠嗅球分离、培养出表达巢蛋白(nestin)，并能分化为神经元、星形胶质细胞和少突胶质细胞的NSC。生后第1天和成年大鼠嗅球的神经球形成率分别为20％～30％和0．1％。嗅球NSC的增殖依赖表皮生长因子(epidermal growth factor，EGF)和碱性成纤维细胞生长因子(fibroblast growth factor—basic，bFGF)，其中EGF的促分裂增殖作用明显优于bFGF。结论从生后第1天和成年大鼠嗅球可以培养出具有自我增殖和多向分化潜能的NSC。     

大鼠嗅上皮神经干细胞的分离及培养

目的：从新生及成年大鼠嗅上皮分离培养嗅上皮神经干细胞（NSC）,研究其增殖及分化特性。方法：采用含10％胎牛血清的DMEM／F12（1：1）培养液,分离、培养生后第3天和硫酸锌原位创伤后6d的成年大鼠嗅上皮NSC,应用间接免疫荧光染色鉴定培养的NSC及分化的特异性神经细胞类型,四甲基偶氮唑盐法（MTT）测定嗅上皮NSC的生长曲线及生长因子的影响。结果：从生后第3天和成年大鼠嗅上皮分离、培养出巢蛋白免疫反应阳性而细胞角蛋白免疫反应阴性,并能分化为神经元和星形胶质细胞的NSC。生后第3天和成年大鼠嗅上皮NSC生存能力的差异无统计学意义（P〉O．05）,细胞克隆形成率为0．05％～0．10％。碱性成纤维细胞生长因子可以显著促进嗅上皮NSC的增殖。结论：从生后第3天和成年大鼠嗅上皮可培养出具有自我复制和多向分化潜能的NSC。     

胚胎大鼠神经干细胞的分离培养和鉴定及标记

目的:探索胚胎大鼠神经干细胞的分离培养、鉴定及标记的方法.方法:分离胚胎大鼠海马组织,用无血清培养技术培养神经干细胞,免疫组织化学荧光技术检测其巢蛋白(Nestin)的表达;5-溴脱氧尿嘧啶(BrDU)掺入试验,并用免疫荧光双标技术观测神经干细胞的增殖状况.采用荧光染料Hoechest33342标记神经干细胞并诱导其分化,用神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)免疫组织化学荧光染色鉴定分化细胞.结果:①获得大量未分化呈巢状悬浮生长的神经干细胞团,并能分化为神经元和神经胶质细胞,且经传代培养8代后仍具干细胞特性;②荧光染料的标记率可达97%,细胞传8代后荧光亮度无明显衰减,并且分化后的细胞核中仍有荧光表达.结论:成功培养出胚胎大鼠神经干细胞,培养出的细胞具有增殖、自我更新及核多潜能分化能力,可分化为神经元及神经胶质细胞;荧光染料的标记可获得较高的标记率,可作为神经干细胞移植实验研究的供体细胞.     

大鼠嗅球神经干细胞培养

目的　建立大鼠嗅球神经干细胞 (neuralstemcells,NSC)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、培养新生第 1天和成年大鼠嗅球NSC ,应用免疫细胞化学方法鉴定培养的NSC及自然分化为特异性神经细胞的类型 ,四甲基偶氮唑盐 (MTT)法测定嗅球NSC的生长曲线。结果　从生后第 1天和成年大鼠嗅球分离、培养出表达巢蛋白(nestin) ,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSC。生后第 1天和成年大鼠嗅球的神经球形成率分别为 2 0 %～ 30 %和 0 1%。嗅球NSC的增殖依赖表皮生长因子 (epidermalgrowthfactor,EGF)和碱性成纤维细胞生长因子 (fibroblastgrowthfactor basic,bFGF) ,其中EGF的促分裂增殖作用明显优于bFGF。结论　从生后第 1天和成年大鼠嗅球可以培养出具有自我增殖和多向分化潜能的NSC。     

大鼠嗅球神经干细胞的超微结构

目的研究体外培养的大鼠嗅球神经干细胞(neuralstemcell,NSC)的超微结构。方法采用添加丝裂原的无血清培养基分离、培养出生后第1天大鼠嗅球NSC,分别于培养后1周,1个月及2个月收集NSC,2.5%戊二醛固定,行扫描电镜及透射电镜观察。结果体外培养的NSC呈圆形或椭圆形,表面有微突起或微绒毛,细胞核浆比例高,稀少的细胞浆中有多少不等的未成熟线粒体、核糖体及高尔基复合体。不同培养时期的神经球内未分化NSC的结构大致相同,均有分裂增殖细胞及少数凋亡细胞。培养1～2个月后出现存在粗面内质网及粗长突起的分化细胞。相邻分化细胞的突起间有缝隙连接。结论体外培养的大鼠嗅球NSC保持原始细胞的形态和结构,并能分化成神经细胞。     

胚胎小鼠听皮层区域神经干细胞的分离培养及鉴定

目的 通过剖腹手术从C57BL/6胎鼠(El4左右)中获得听皮层区域组织,进行神经干细胞体外培养和分化鉴定,探索新的自体NSC移植治疗来源.方法 选取孕14天左右的C57BL/6小鼠,剖腹取出胎鼠,分离听皮层区域组织,在体外无血清培养得到NSC,用免疫荧光细胞染色法进行NSC特异性标记物(Nestin)、增殖能力(Br...     

Upregulation of neural growth-associated protein and neural cell adhesion molecule in mouse olfactory epithelium and axons after unilateral removal of the olfactory bulb

The expression of synaptosomal-associated protein (SNAP-25), neural growth-associated protein (GAP-43) and neural cell adhesion molecule (NCAM) were studied in mouse olfactory cells and axons for 2 weeks following unilateral bulbectomy. The olfactory cells and axons in the control olfactory epithelium were positive for SNAP-25 but levels decreased in the atrophic olfactory epithelium 3 days after bulbectomy. There was no expression of SNAP-25 in the olfactory epithelium on the bulbectomy side 7 days after bulbectomy, indicating that this protein may be a good marker for the degeneration of olfactory cells. The expression of NCAM was still found in the atrophic olfactory epithelium at 7 days after bulbectomy, while the expression of NCAM in the olfactory epithelium of the bulbectomy side was stronger than that on the control side at 14 days after bulbectomy. The expression of GAP-43 in the olfactory axonal bundles of the bulbectomy side at 3 and 4 days after bulbectomy was stronger than that on the control side. These results suggest that upregulation of NCAM may be related to the regeneration of the olfactory cells, with upregulation of GAP-43 probably playing a role in axonal regeneration after bulbectomy. Received: 6 January 1998 / Accepted: 22 April 1998     

High-dose glucocorticoids inhibit proliferation of rat olfactory epithelium

Glucocorticoids (GCs) are commonly prescribed for treatment of olfactory dysfunction. However, the effects of GCs on olfactory epithelium are not well known. We investigated the effects of high-dose GCs on proliferating cells of olfactory epithelium. Five adult male rats (300 g) received a single daily subcutaneous dose of vehicle containing 0.3 mg dexamethasone (DEX) for 9 days (DEX+ group), and a control group received vehicle alone (DEX- group). We compared sections from the two groups for numbers of Ki67-positive cells. The mean number of Ki67-positive cells per 500 olfactory epithelial cells was 9.6 for the DEX+ group and 58 for the DEX- group (significant difference). We conclude that high-dose GC suppressed proliferation of olfactory epithelium. We suggest that high-dose GC suppresses cytokines and growth factors, resulting in secondary suppression of proliferating ability.     

Culture and characterization of rat middle-ear epithelium

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.