外伤性嗅觉障碍大鼠嗅黏膜的组织学变化

目的 构建大鼠外伤性嗅觉障碍模型,观察不同时间点嗅黏膜组织学变化.方法 神经切断组(40只)和对照组(20只)大鼠均在显微镜下暴露左侧嗅球,沿筛板切断神经组切断大鼠左侧嗅神经.采用嗅觉诱发电位(olfactory evoked potentials,OEPs)和神经示踪验证造模效果.术后1天、5天、2周、3周、6周处理大鼠,处理前1天每组各取2只大鼠经鼻腔滴注辣根过氧化物酶(horseradish peroxidase,HRP).嗅黏膜及嗅球冰冻切片后观察嗅上皮的厚度、细胞数量的变化以及嗅神经的连续性,并且行免疫组化观察嗅上皮中的新生嗅感觉神经元(olfactory receptor neurons,ORNs).结果OEPs及神经示踪证实手术方法可以完全切断嗅神经.术后1天,切断侧黏膜中ORNs出现凋亡,两组大鼠双侧嗅上皮厚度和细胞数量比值无明显变化.5天时切断侧嗅上皮中细胞数量减少,上皮厚度变薄,嗅球中无HRP标记纤维.术后2、3周大鼠嗅球中出现蓝色标记,嗅上皮厚度和细胞数量逐渐增加,但仍然与对照组有差异,此时嗅上皮中出现大量的新生ORNs.经过6周的恢复,嗅上皮厚度及细胞数量基本恢复至对照组水平,嗅上皮中有较多的新生ORNs,其轴突与嗅球重新建立神经联系,但是上皮中仍然有一定数量的凋亡细胞.结论 嗅神经切断术可以作为制作外伤性嗅觉障碍的可靠方法;由于ORNs具有再生能力,大鼠嗅神经切断后嗅黏膜在一定时间内基本恢复至正常水平.     

白血病抑制因子与嗅感觉神经元再生

哺乳动物的嗅感觉神经元(olfctory receptor neurons,ORNs)均具有终生可再生的特性.嗅上皮内ORNs的数目之所以能维持在一个相对恒定的水平,是因为ORNs的死亡和再生处于动态平衡状态.有多种细胞因子参与维持这种动态平衡,而白血病抑制因子(leukemia inhibitoryfactor,LIF)是近年研究得比较多的一种细胞因子.本文在介绍LIF的结构、分布和信号转导机制后,综述近年来开展的有关LIF与ORNs再生的研究,并分析其在这一过程中可能发挥的作用.     

白血病抑制因子与嗅感觉神经元再生

哺乳动物的嗅感觉神经元(olfactory receptor neurons，ORNs)均具有终生可再生的特性。嗅上皮内ORNs的数目之所以能维持在一个相对恒定的水平，是因为ORNs的死亡和再生处于动态平衡状态。有多种细胞因子参与维持这种动态平衡，而白血病抑制因子(leukemia inhibitory factor，LIF)是近年研究得比较多的一种细胞因子。本在介绍LIF的结构、分布和信号转导机制后，综述近年来开展的有关LIF与ORNs再生的研究，并分析其在这一过程中可能发挥的作用。     

嗅感觉神经元的再生调控及与嗅觉障碍的关系

嗅感觉神经元（olfactory receptor neurons，ORNs）在嗅觉中有重要的作用，且能够不断更新。ORNs的干细胞存在于嗅上皮的球形基底细胞中，ORNs的前体分为三个阶段，每个阶段都是ORNs产生的重要调控点。而对ORNs的调控分子信号主要为两个多肽类生长因子的超家族，这些分子信号对ORNs的再生和保持嗅上皮中适当数量的ORNs都有重要作用。了解ORNs再生的调控机制，研究促进ORNs的再生．可为治疗嗅觉障碍提供一些有效的方法。     

嗅感觉神经元凋亡的信号转导途径及基因调控

哺乳动物嗅上皮内衰老和受损伤的嗅感觉神经元(olfactory receptor neurons,ORNs)以凋亡的方式被机体清除,ORNs凋亡的发生、发展是在基因精确调控下通过两条信号转导途径来完成的.与凋亡相伴行的足ORNs的再生,二者的平衡确保了嗅上皮层ORNs的新旧更替和嗅觉功能的维持.本文通过复习ORNs凋亡和再生的相关文献,阐述ORNs的损伤因素及其凋亡的信号转导途径与基因调控.     

嗅上皮的组织块培养法

目的建立并优化组织块法嗅觉受体神经元（olfactory receptor neurons,ORNs）原代培养体系。方法以鼠尾胶原为培养底物,取新生小鼠鼻中隔和筛甲的嗅上皮进行原代培养。观察包括ORNs在内的各种细胞的形态和分布特点,经免疫组化和扫描电镜证实本原代培养体系内各种细胞的属性以及ORNs的分化程度。结果组织块培养法所得为一混杂的培养体系,除ORNs外还有基底细胞、嗅鞘细胞、纺锤形细胞及成纤维细胞等。其中基底细胞和纺锤形细胞在形态和免疫表型上均具有ORNs前体细胞的特点。优化培养体系后,可得到分化成熟的ORNs并可在体外稳定存活9天以上。结论本培养方法避免了使用消化酶,可以稳定的得到分化成熟的ORNs。     

嗅感觉神经元再生的研究现状

脊椎动物的嗅感觉神经元(olfactory receptorneurons,ORNs)具有独特的终生可持续再生的能力。本文在复习嗅黏膜的结构后,将从研究ORNs再生的手段、ORNs的再生过程和影响这一过程的分子信号三方面复习文献,对这一领域的研究现状进行综述。     

放射线损伤对小鼠嗅上皮细胞凋亡及再生的影响

目的研究放射线损伤后小鼠嗅上皮细胞凋亡和再生情况,探讨放射治疗后嗅觉障碍的致病机制。方法以4Gy的X射线照射小鼠鼻部,建立放射线损伤小鼠嗅黏膜的动物模型分别于照射前及照射后第1,3,6,12,60天分批处死动物取材;TUNEL法检测嗅上皮中细胞凋亡,BrdU掺入免疫组化检测基底细胞再生。结果放射线损伤后小鼠嗅上皮凋亡细胞显著增多（P〈0.01）,基底细胞再生也相应增多（P〈0.01）,但再生细胞少于凋亡细胞（P〈0.01）。结论放射线损伤可促进嗅上皮细胞凋亡及基底细胞再生,但再生细胞少于凋亡细胞,二者失衡可能是放射治疗后嗅觉障碍的致病机制之一。     

流感病毒感染后小鼠嗅感觉神经元的凋亡与再生

目的 研究病毒感染后小鼠嗅感觉神经元的凋亡和再生，探讨病毒感染后嗅觉障碍(PVOD)的发病机制。方法 经前鼻孔接种流感病毒，感染实验动物的鼻腔；TUNEL技术检测嗅感觉神经元凋亡；BrdU掺入单克隆抗体标记技术检测基底细胞增殖。结果 ①流感病毒感染后嗅感觉神经元凋亡显著增多，恢复期凋亡细胞逐渐减少。②基底细胞增殖早期有所增多，随后减少至和凋亡细胞相当的水平。结论 流感病毒感染可有效地诱导嗅感觉神经元凋亡，并促进基底细胞增殖；基底细胞增殖不足以补充嗅感觉神经元凋亡，导致嗅感觉神经元总数减少。     

凋亡相关蛋白Bcl-2和Bax在慢性鼻及鼻窦炎伴嗅觉障碍患者嗅黏膜中的表达

目的研究Bcl-2和Bax在慢性鼻及鼻窦炎（CRS）伴嗅觉障碍患者嗅黏膜中的表达,探讨其对嗅觉神经元（olfactory receptor neurons,ORNs）凋亡的调节作用。方法采用康涅狄格化学感受临床研究中心所采用的嗅觉检查法——CCCRC（Connecticut ChemosensoryClinical Research Center）对46例行功能性鼻内镜手术的患者进行嗅觉评分并分组：A组,CRS伴嗅觉障碍25例;B组,CRS不伴嗅觉障碍10例;C组,单纯鼻中隔偏曲行鼻中隔矫正术11例。免疫组织化学方法检测Bcl-2、Bax在3组患者嗅黏膜中的表达。结果在ORNs中,A组Bcl-2和Bax表达显著高于B组（q=3.24、4.29,P均〈0.05）和C组（q=8.56、12.99,P均〈0.01）,A组Bcl-2/Bax比值显著低于B组（q=3.76,P〈0.05）和C组（q=6.67,P〈0.01）;在基底细胞中,Bcl-2在3组表达无显著差异（q=0.68、0.69、1.06,P均〉0.05）,Bax在A组的表达显著高于B组和C组（q=9.54、11.98,P均〈0.01）,A组Bcl-2/Bax比值显著低于B组和C组（q=5.48、9.14,P均〈0.01）;在A、B、C 3组ORNs和基底细胞中,Bcl-2/Bax比值与嗅觉评分均呈正相关（rA分别为0.5631、0.8926,rB分别为0.5700、0.7991、rC分别为0.5694、0.8121,P均〈0.01）。结论细胞凋亡参与了CRS伴嗅觉障碍患者ORNs的减少,Bcl-2和Bax在此过程中起了重要的调控作用,Bcl-2/Bax比值决定细胞是否凋亡。     

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.     

变应性鼻炎嗅觉障碍大鼠嗅感受神经元的实验研究

目的　通过锰增强磁共振成像技术（manganese-enhanced magnetic resonance imaging,MEMRI）观察变应性鼻炎（AR）嗅觉障碍大鼠嗅觉传导通路的改变,并用病理学方法观察嗅黏膜形态结构的改变和相关免疫组织化学变化,来探讨AR对嗅黏膜感受神经元的影响。方法　40只SD大鼠,随机分为实验组（30只）和对照组（10只）。实验组应用卵清蛋白致敏并激发SD大鼠,应用埋藏食物小球实验评估AR大鼠嗅觉功能,建立AR嗅觉障碍的大鼠模型。ELISA法测定并比较血清IgE水平。MEMRI观察大鼠嗅球中锰增强的对比显影。HE染色及免疫组化法观察嗅黏膜的组织形态学变化及嗅标记蛋白表达的变化。结果　对成功建模的AR大鼠进行嗅觉功能评估,40%（12/30）的AR大鼠在AR的基础上伴有嗅觉障碍。MEMRI示对照组大鼠嗅球锰增强显影显著,AR伴嗅觉障碍组大鼠嗅球几乎无锰增强显影,AR不伴嗅觉障碍组大鼠嗅球有部分锰增强显影。AR伴嗅觉障碍组大鼠嗅黏膜的上皮层明显变薄,阳性的嗅感受神经元的层数减少,与不伴嗅觉障碍组和对照组相比差异存在统计学意义（P 均<0.05）。结论　通过卵清蛋白致敏激发可以成功建立AR嗅觉障碍的大鼠模型。AR可导致嗅黏膜感受神经元的变化,并由此导致嗅觉传导通路的改变,有可能是AR引起嗅觉障碍的发病机制之一。     

嗅神经切断对小鼠嗅感觉神经元的影响

目的分析嗅神经切断对小鼠嗅感觉神经元（olfactory receptor neurons，ORN）凋亡情况的影响，并探讨这一造模方法的可靠件。方法取2月龄C57小鼠33只，随机分组后，实验组小鼠行嗅神经切断术，通过辣根过氧化物酶（horseradish peroxidase，HRP）顺行神经示踪验证造模成功与否。以脱氧核苷酸转移酶介导的牛物素dUTP缺口末端标记技术（TdT mediated deoxyuridine triphosphate—biotin nick end labelling，TUNEI。）观察术后8h、2d、3d和5d嗅上皮中ORN的凋亡情况，同时在蛋白和mRNA水平观察成熟ORN的特异性标记蛋白——嗅标记蛋白（olfactory marker protein，OMP）在嗅上皮巾的表达情况。结果嗅神经切断术后嗅球中无HRP标记。TUNEL和OMP阳性反应发生于ORN，嗅神经切断术后TUNEL阳性细胞数显著增多，并于术后第2天达到高峰，与此同时OMPmRNA的表达水平开始显著下降，并住术后第5天降至更低，嗅上皮的厚度也相应变薄。结论本实验所采取的造模方法可以较可靠地切断小鼠的嗅神经，并造成小鼠嗅上皮中ORN的同步凋亡。     

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.     

Lipopolysaccharide-induced apoptosis of olfactory receptor neurons in rats

CONCLUSION: Daily intranasal perfusion of lipopolysaccharide (LPS) for 14 days in rats induced apoptosis of olfactory receptor neurons (ORNs) over >3 but <7 days. OBJECTIVES: Smoking is one of the factors causing olfactory dysfunction. LPS is a major glycolipid component of the gram-negative bacterial cell wall and an active component of cigarette smoke. We studied whether LPS is one of the causes of tobacco-induced olfactory dysfunction by examining apoptosis in the olfactory epithelium after local exposure to LPS. MATERIALS AND METHODS: Rats received intranasal instillation of LPS or saline. Histochemical changes in the olfactory epithelium were examined using antibodies against single-stranded DNA, Bcl-2, Bax, and Caspase-3. We used different concentrations of LPS to examine the dose dependency and observed changes in the olfactory epithelium for a week after exposure cessation to see the duration of the effect of smoking. RESULTS: We found that numbers of cells positive for ssDNA, Bcl-2, Bax, and Caspase-3 were increased on the exposed side. The number of ssDNA-positive cells reached a maximum on the first day and decreased to normal levels on the seventh day after cessation of exposure.     

The dynamics of precursor cells in the olfactory epithelium of juvenile and adult guinea pigs

The dynamics of precursor cells in the olfactory epithelium of juvenile and adult guinea pigs were examined by immunohistochemical double staining using bromodeoxyuridine (BrdU), the neural cell adhesion molecule (N-CAM) and the protein gene product 9.5 (PGP9.5). Expression of apoptotic cells in the olfactory epithelium with the use of the TdT-mediated dUTP biotin nick end labeling (TUNEL) method was also observed. BrdU was given to healthy guinea pigs at the ages of 2 weeks and 6 months old. Tissue specimens were serially collected 1 h to 28 days after administration. BrdU-labeled cells were seen above the basal cell layer after 1 h and migrated to the middle layer of the olfactory epithelium, after 1 day in juveniles and 5 days in adults with expression of N-CAM. PGP9.5 was observed in BrdU-labeled cells after 5 days in juvenile guinea pigs and 7 days in adult. At 14 days after administration, BrdU-labeled cells in the epithelium appeared to decrease. However, a few of these cells were recognized above the basal cell layer after 28 days. The number and location of TUNEL-positive cells did not significantly differ between the juvenile and adult olfactory epithelium. Therefore, we conclude that the division speed from stem cells in juveniles is faster than that in adults, and apoptosis is unaffected by aging in the normal olfactory epithelium.     

倍他米松对大鼠离体嗅感觉神经元cAMP的影响

目的 观察倍他米松对大鼠嗅感觉神经元内环磷酸腺苷（cyclic adenosine monophosphate，cAMP）的影响，探讨糖皮质激素治疗嗅觉障碍的可能机制。方法取10只大鼠，提取嗅感觉神经元细胞膜蛋白，加入终质量浓度为0．1mg／ml和1．0mg／ml的倍他米松预孵育，采用酶联免疫吸附实验观察不同孵育时间点（5min和30min）环核苷酸门控通道（cyclic nucleotide-gated channel，CNG通道）配体cAMP生成量的变化。结果0．1mg／ml和1．0rag／ml倍他米松可提高大鼠嗅感觉神经元中cAMP生成量，同一时间点两个浓度相比差异具有统计学意义（P值均〈0．05）；倍他米松预孵育5min后即引起cAMP浓度的快速升高，直到30min仍维持较高水平，同一浓度两个时间点比较差异无统计学意义（P值均〉0．05）。结论倍他米松可使嗅感觉神经元内cAMP生成量增多，且1．0mg／ml倍他米松的作用大于0．1mg／ml的倍他米松。糖皮质激素对嗅觉障碍的治疗作用可能与嗅觉信号转导过程中腺苷酸环化酶-环磷酸腺苷通路强化有关。