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。     

大鼠嗅上皮神经干细胞培养及鉴定

胚胎及成年神经干细胞(neural stem cell，NSC)的成功培养为中枢神经系统疾病治疗带来了新的策略。从颅外组织如嗅上皮获取：NSC可能是自体NSC移植的理想途径。研究表明，成年人及小鼠的嗅上皮中含有NSC。本研究观察新生及成年大鼠嗅上皮NSC的增殖及分化特性，为嗅上皮NSC的进一步研究提供参考。     

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。     

不同发育时期嗅上皮的演变

研究不同发育时期大鼠嗅上皮的形态学变化。Spragne－Dawley大鼠按不同发育时期分为四组：胚胎19天鼠（E_19），出生日鼠（P_o），生后10天鼠（P_10），成年鼠（3个月，250g～350g）。采用三种研究方法：①取大鼠鼻中隔嗅粘膜，常规处理后Epon包埋，半薄切片（厚1μm），光镜下观察嗅上皮的厚度和细胞层数；②取P1鼠、P_8鼠和成年鼠，将逆行示踪剂──荧光金（FG）注入右侧嗅球，三天后以同法取嗅上皮，经固定、包理、染色后显微照像，计算FG标记的成熟感受器细胞数和百分率（标记细胞数／总感受器细胞数）；③取P1鼠和成年鼠，切除右…     

鼻科学

20050442大鼠嗅球发育过程中的凋亡与增殖/陈福权…//第四军医大学学报.2004,25(20).1850～1852目的:探讨大鼠嗅球发育过程中细胞凋亡和增殖的变化规律。方法:胚胎14天,新生1天(postna talday,P1),P20及成年大鼠各10只,取嗅球以40g/L多聚甲醛固定,石蜡包埋,连续切片。采用TUNEL和免疫荧光染色方法检测大鼠嗅球中凋亡细胞和增殖细胞核抗原(PCNA)免疫反应阳性细胞的分布。结果:在胚胎14天和P1大鼠嗅球中,凋亡细胞广泛分布而PCNA免疫反应阳性细胞主要分布在室管膜周围区域,凋亡细胞和PCNA免疫反应阳性细胞在p20和成年大鼠嗅球中主要分…     

小鼠嗅上皮增殖细胞和表皮生长因子受体的免疫组化定位

即使在成年动物中 ,嗅觉细胞不断发生增殖、分化、死亡 ,随年龄老化嗅上皮发生萎缩和嗅功能衰退。嗅功能与年龄相关的减退可能与炎症过程、鼻粘膜清洁功能减退、嗅球体积减少、嗅皮层树突衰退有关 ,认为年龄相关嗅功能减退的可能机理是嗅上皮萎缩 ,而这种萎缩与细胞增殖减退有关。增殖细胞核抗原 ( PC-NA)抗体已经被广泛用于各种正常和肿瘤组织中检测扩增核酸。因此 ,该作者用 PCNA抗体和抗人 EGFRs抗体的免疫组化方法检测小鼠嗅上皮增殖细胞和表皮生长因子受体的年龄变化。小鼠分为三组 :胚胎鼠 ( 14天、16天 )、初生鼠 (出生后 1天、…     

转基因诱导新生大鼠耳蜗大、小上皮嵴细胞分化为毛细胞样细胞实验

目的通过转染Hathl基因诱导大鼠耳蜗大上皮嵴细胞（greater epithelial ridge,GER）和小上皮嵴细胞（1esser epithelial ridge,LER）分化为毛细胞样细胞。方法取出生后第l天的大鼠耳蜗,利用机械分离和酶消化相结合的方法分别分离出纯的GER和LER细胞,并转染Hathl基因,培养后做免疫组化染色观察。结果GER和LER体外培养并转染Hathl基因后,免疫组化检测显示肌球蛋白（myosinVIIa）染色阳性,表达毛细胞的特异标记物。提示GER和LER已分化为毛细胞样细胞。结论GER和LER细胞作为毛细胞前体细胞可以实现体外培养,转染Hathl后．可分化为毛细胞样细胞。     

红景天素上调大鼠嗅球成纤维细胞生长因子及其受体机制的探讨

目的：明确大鼠嗅球中成纤维细胞生长因子（FGF）合成、分泌和作用的靶细胞及其与受体（FGFR）表达的相关性；探讨中药红景天素抗大鼠嗅球衰老的信号传导机制。方法：取Wister大鼠青年对照组；老龄对照组和老龄红景天素用药组各10只，3个月后断头处死，取嗅球，以原位杂交和免疫组化方法分别检测FGFmRNA和FGF、FGFR蛋白在大鼠嗅球中的表达。结果：随着年龄的增长，大鼠嗅球中FGFmRNA、FGF、FGFR的表达明显减少；青年对照组和老龄对照组差异显著（P＜0．01）；老龄红景天素组FGFmRNA、FGF、FGFR明显高于老龄对照组（P＜0．05）。并且FGFR与FGF的表达呈明显的正相关。结论：大鼠嗅球组织的生长，发育与成纤维细胞生长因子及其受体信号传导密切相关；推测红景天素主要通过诱导大鼠嗅球FGF的表达，上调其受体FGFR的信号传导途径，发挥其对嗅球的抗衰老作用。     

二甲胺四环素对嗅感觉神经元凋亡的保护作用

目的：观察二甲胺四环素对慢性鼻窦炎大鼠嗅感觉神经元（OSNs）凋亡的保护作用。方法：将大鼠随机分为对照组（A组）、鼻窭炎造模组（B组）和鼻窦炎加二甲胺四环素组（C组）,于不同时间取材,用苏木精-伊红染色观察嗅上皮厚度及上颌窦黏膜病理变化,用免疫组织化学染色检测Caspase-3表达阳性细胞。结果：苏木精-伊红染色镜检发现上颌窦黏膜病理学改变：A组明显轻于B组和C组,B、C两组嗅上皮厚度差异有统计学意义（P〈0．05）,免疫组织化学镜检发现B、C两组Caspase-3阳性细胞表达差异有统计学意义（P〈0．05）。结论：二甲胺四环素能抑制鼻窦炎大鼠模型Caspaser-3的表达,可能成为治疗嗅觉障碍的有效药物。     

C57BL／6-gfp小鼠嗅球神经干细胞的培养鉴定及耳蜗核移植

目的建立C57BL／6－gfp小鼠嗅球神经干细胞体外培养的方法,并初步应用于大鼠耳蜗核定位移植。方法培养C57BL／6－gfp小鼠胚胎嗅球神经千细胞,传代并进行分化实验及鉴定后将其立体定位注射于大鼠耳蜗核。结果所培养的神经干细胞生长良好,可稳定传代,能够分化为3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小鼠胚胎嗅球神经干细胞可稳定传代并可以作为荧光标记细胞进行移植实验。     

大鼠神经干细胞生长及增殖规律的体外研究

目的:体外无血清条件下培养胚鼠神经干细胞,观察其生长及分化情况.方法:取孕16～18 d SD系大鼠的胚胎海马组织,在含EGF、bFGF和B27的DMEM/F12培养基中培养,电子显微镜下观察其增殖分化过程,并通过免疫荧光方法鉴定分化后的细胞类型.结果:神经干细胞在无血清培养基中生长旺盛,8 d左右就可以形成胞体透亮、折光性好的干细胞球,分化后的细胞显示NSE、GFAP免疫阳性.结论:无血清培养条件下神经干细胞生长良好,而在含血清的培养基中可以分化为神经元和星形胶质细胞.     

Blocking P2X receptors can inhibit the injury-induced proliferation of olfactory epithelium progenitor cells in adult mouse

Objective

The olfactory epithelium (OE) is unusual for its remarkable regenerative capacity and sustained neurogenesis of olfactory receptor neurons (ORNs) throughout adult life. Regeneration of ORNs is accomplished by basal cells in the OE, including stem cells and progenitor cells. Although there is considerable knowledge about the roles of OE basal cells in ORN turnover, the molecular mechanism that regulates the proliferation and differentiation of adult OE basal cells is not fully understood. As intercellular signaling molecules, purines have been reported to meditate proliferation, differentiation and migration of many kinds of neural stem cells. However, it is still unclear whether ATP, which could be released by injured ORNs, plays a role in regulating neurogenesis in ORN turnover.

Methods

RT-PCR and immunohistochemistry were used to detect the expression of ionotropic purinergic receptors-P2X receptors in adult mouse OE. By using the olfactory bulbectomy model and in vivo administration of P2X receptors antagonists, the function of P2X receptors in regulating the proliferation of OE progenitor cell was evaluated.

Results

We found that basal cells in the adult mouse OE express functional P2X receptors, and blocking the activities of P2X receptors can significantly inhibit the injury-induced proliferation of OE basal cells.

Conclusion

Our research provides evidence in support of the hypothesis that purinergic signaling can serve as a paracrine signal in regulating the neurogenesis of OE in adult mouse.     

The effect of basic fibroblast growth factor on the regeneration of guinea pig olfactory epithelium

Olfactory receptor cells are widely thought to regenerate after degeneration and also thought to show turnover in normal circumstances in animal olfactory epithelium. The identity of the factor that controls proliferation and differentiation of olfactory receptor cells is a very important problem that has yet to be resolved. In this study, the mitogenic effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on olfactory receptor cells in guinea pig olfactory epithelium was examined. The intraperitoneal injection of 1,000 ng bFGF/day for 14 days increased the cells in proliferation detected by immunostaining with proliferating cell nuclear antigen (PCNA), while neither EGF nor low-dose bFGF had any effect. These results support the idea that an adequate dose of bFGF plays an important role in the neurogenesis in the olfactory epithelium. Further study is needed to clarify the efficacy of bFGF in the damaged olfactory epithelium, but bFGF may provide a therapeutic option for olfactory disturbances caused by complete or partial loss of olfactory receptor cells. Received: 3 October 2001 / Accepted: 10 October 2001     

豚鼠胚胎神经干细胞的分离培养、鉴定及标记

目的：从胚胎豚鼠端脑组织中分离培养神经干细胞，为神经干细胞移植治疗感音神经性耳聋的实验研究创造条件。方法：分离胚胎豚鼠端脑组织，用含碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)无血清培养技术培养神经干细胞，免疫组化技术检测其巢蛋白(Nestin)的表达及细胞分化后胶质纤维酸性蛋白(GFAP)的表达。采用荧光染料Hoechst33342标记神经干细胞。结果:从胚胎豚鼠端脑组织分离出的细胞中可获得呈集落样生长的神经干细胞团，并能表达Nestin和分化成神经元和神经胶质细胞；荧光染料的标记效率可达96．7％，细胞传代6次后荧光亮度仍无明显衰减。结论:从胚胎豚鼠端脑分离的细胞具有明显的增殖能力及多分化潜能，荧光染料的标记可获得较高的标记效率，可作为神经干细胞移植治疗感音神经性耳聋实验研究的供体细胞。     

神经元核心抗原在小鼠嗅球和嗅上皮发育中的表达

目的：探讨神经元核心抗原（NeuN）在嗅球和嗅上皮发育过程中的表达特性和意义。方法：在小鼠胚胎发育9．5、11．5、14．5、17．5d,出生当天和3个月成年个体的头部切片标本中,以免疫荧光染色方法检测NeuN的表达。结果：刚出生小鼠嗅球内层状结构尚不明显,NeuN表达于嗅球周边区域。在3个月大成年小鼠,嗅球的层状结构已清晰可见,NeuN表达阳性的成熟神经元主要聚集于接近嗅球中心的颗粒细胞层。位于嗅上皮的双极嗅感觉神经元在小鼠胚胎和成年个体中均未见NeuN表达。结论：NeuN通常被认为表达于几乎所有部位的成熟神经元。但在嗅球和嗅上皮中,NeuN只表达于成熟嗅球中的颗粒细胞层。小鼠出生到发育为成熟个体的过程中,NeuN表达阳性的成熟神经细胞由周边逐渐向嗅球中心迁移,可能与气味模式的形成有关。     

c-myc在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化

目的 通过检测c-myc基因在大鼠耳蜗组织发育及前体细胞分化过程中的表达情况,探讨其在哺乳动物耳蜗发育中的作用.方法 ①取E10、E15、P1、P7和P14的SD大鼠耳蜗组织,应用RT-PCR、Western blot的方法检测c-myc在大鼠耳蜗组织发育过程中的表达情况.②取耳蜗前体细胞和分化7天后的分化细胞,用RT-PCR、免疫细胞化学染色和Western blot的方法检测c-myc在耳蜗前体细胞分化过程中的表达情况.结果 从胚胎至出生后的耳蜗发育过程中,c-myc表达量呈现逐渐下降趋势;在前体细胞的分化过程中,c-myc的表达量也呈现下降趋势.结论 c-myc可能参与调控大鼠耳蜗组织的发育及前体细胞的分化.     

Maintenance of regional difference in cellular composition of neurospheres derived from adult mouse olfactory bulb

Neurospheres are potentially good tools to study olfactory neurogenesis. For this purpose, it is necessary to characterize neurospheres derived from the olfactory system. This study is aimed at identifying the regional difference of cellular composition within neurospheres derived from the adult mouse olfactory bulb, and studying whether these characteristics are maintained throughout the long-term culture. Neural cells were obtained from the olfactory bulbs of 8-week-old Balb/c mice and the dissociated cells were cultured in serum-free media containing epidermal growth factor and basic fibroblast growth factor to form neurospheres. These neurospheres were subjected to characterization by confocal microscopy after immunofluorescence staining with nestin, proliferating cell nuclear antigen (PCNA), neuronal cell adhesion molecule (NCAM), glial fibrillary acidic protein (GFAP), O4, and olfactory marker protein (OMP). Confocal microscopy showed that nestin-reactive cells, which are stem cell-like cells, were present at the periphery of neurospheres and PCNA-reactive cells undergoing proliferation were found throughout the neurospheres. Neuronal cell markers, NCAM-reactive cells, were observed at the center of neurospheres while glial cell markers, GFAP- or O4-reactive cells, were present at the periphery of neurospheres. No cells were found to be reactive for OMP, a differentiated olfactory neuron marker. This regional distribution of cells in neurospheres was maintained through 17 passages for a year. The neural lineage of cells showed different distribution within neurospheres and this localization was preserved throughout the culture period. These findings in the neurosphere culture system of the olfactory bulb may help to study olfactory neurogenesis.     

Immunohistochemical localization of proliferating cells and epidermal growth factor receptors in mouse olfactory epithelium

We investigated age-related changes in proliferating cells and epidermal growth factor receptors (EGFRs) in mouse olfactory epithelium using an immunohistochemical method with the antiproliferating cell nuclear antigen (PCNA) antibody and the antihuman EGFRs antibody. Many PCNA-positive cells occurred in the surface and basal layers of the olfactory epithelium in the embryonal and neonatal mice. However, only some PCNA-positive cells occurred in the basal layer of adult mice, and only a few presented in the basal layer of aged mice. EGFRs were observed in all layers of the olfactory epithelium at the embryonal and neonatal stages, but were not identified in the olfactory epithelium at the adult or aged stages. We believe that a decrease in EGFRs in the olfactory epithelium induces the inhibition of cell proliferation, with the resultant atrophy of the olfactory epithelium.