【作者】 史保院；

【导师】 史丽；

【作者基本信息】 山东大学 ， 耳鼻咽喉， 2008， 硕士

【摘要】 [研究背景]嗅觉是哺乳动物的重要感觉器官之一,它同视觉、听觉一样,是人体捕获外界信息的特殊装置。人类的嗅觉具有辨别气味、增进食欲、识别环境、报警等作用。嗅觉还通过中枢神经系统影响人的情绪、调节生命周期[1]。对一些特殊职业,如香精师、美食家、侦察员、医师、公安消防人员等,灵敏的嗅觉更是必不可少。嗅神经元是唯一暴露在外界环境中的神经元容易受到外界的损伤。FrankME[2]等报道人类永久性嗅觉障碍发病率约1.2%,暂时性嗅觉障碍发病率约62.4%,由于缺乏客观检查方法和发生机制不明等原因,嗅觉障碍一直缺乏有效的治疗方法。随着人们生活水平的提高,生活质量有更高的要求,嗅觉越来越引起重视。本实验通过对大鼠嗅感觉上皮发育过程的组织化学及大鼠嗅上皮细胞凋亡的研究以期对嗅觉障碍的发生及治疗提供一定的理论依据。[研究目的2免疫组化方法检测神经特异性烯醇酶(NSE)及嗅细胞标记蛋白(OMP)在胎鼠嗅上皮发育过程中的表达,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术(TUNEL)检测胎鼠嗅上皮发育过程中的细胞凋亡,以揭示哺乳动物胚胎期嗅上皮的发生、发育、成熟及凋亡过程。[研究方法]取大鼠不同孕期(E13-E21)胚胎鼻部标本,免疫组化方法检测神经特异性烯醇酶(NSE)及嗅细胞标记蛋白(OMP)在不同胎龄胎鼠嗅上皮中的表达及其规律。TUNEL方法检测其嗅上皮细胞凋亡,并计算细胞凋亡指数(AI)。统计学方法数据以x±s表示,采用spss10.0统计软件处理,各组间平均数比较采用非参数秩和检验,P＜0.05为具有统计学意义。[结果]大鼠E13天胎鼠嗅上皮中即有神经特异性烯醇酶表达(NSE),细胞数量多,细胞排列尚不规律,没有形成典型的柱状上皮形态。随胎龄增加阳性细胞数量增加,排列规律,细胞形态更接近双极细胞。E14-E21天胎鼠,在嗅上皮中均可见大量阳性细胞。E13天胎鼠嗅上皮中未见OMP阳性表达细胞。在大鼠E14天胎鼠,嗅上皮中开始出现嗅细胞标记蛋白(OMP)阳性细胞,数量少散在分布,随胎龄增加阳性细胞数量增多。至E17天达高峰,以后渐趋稳定。在E13天胎鼠嗅上皮中可见细胞凋亡,细胞凋亡数量少,E13-E15天胎鼠嗅上皮中细胞凋亡数量较稳定,至E16天胎鼠嗅上皮中凋亡细胞数量明显增加,达到高峰,E18天-E21天胎鼠嗅上皮中凋亡细胞数量逐渐减少渐趋于稳定。E16天胎鼠嗅上皮中凋亡指数与其他天数之间P＜0.05,差异有显著性。其他组间P＞0.05。[结论]嗅细胞标记蛋白(OMP)与神经特异性烯醇化酶(NSE)检测的联合应用可反应嗅上皮中嗅细胞发生、发育及成熟过程。大鼠胚胎发育过程中,胚胎中期嗅上皮中已有发育成熟的嗅神经元,胚胎后期嗅上皮已基本发育成熟,大鼠在出生时已具有一定的嗅功能。在嗅上皮发育过程中存在细胞凋亡,细胞凋亡在嗅上皮发生伊始(E13天)即已出现,是一生理过程,E16天出现一高峰,与嗅上皮和嗅球建立突触联系的时间相吻合,可能是两者相互作用的结果,提示细胞凋亡在嗅觉发育过程中对嗅细胞数量的稳定起重要作用。更多还原

【Abstract】 Background:The olfactory is the important sensory organ for mammalian,as well as the visual sense,the auditory sense.It is a special structure for human to capture surrounding message.It can indentify the odour,discern circumstance and promote appetite and so on.Otherwise,it can affect human emotion and adjust life cycle by central nervous system.The sensitive olfactory is absolutely necessary to special occupation,such as the gastrologist,doctor,fire-fighter etc.Then,the olfactory recetor neurns are uniqueness neurns which expose to surrounding and facility to be hurt.Frank ME reported that the morbility of the permance dysosmia is 1.2%.The temporality dysosmia is 62.4%.As the objectively examination is deficiency and the genesis mechanism of dysosmia is not clear.The dysosmia is not cured effectively.This research will utilize immunohistochemical method and the Tdt-mediated dUTP nick end-labeling techniques to detect the development of the mouse olfactory epithelium.It perhaps provides a new path to treat the dysosmia.Object:This research is expected to detect and compare the expression and the value of the neuro-special endolase(NSE),the olfactory marker protein(OMP)and the apoptotic cells in the mouse olfactory epithelium of the different embryomic stages. The immunohistochemical method and the Tdt-mediated dUTP nick end-labeling techniques(TUNEL)were used.we expect to explore the developmental process of the mammal olfactory epithelium.Method:Embryonic mice were sacrificed by decapitation in defferent embryomic stages.The embryonic tissues were dissected out and fixed by immersion in 4% paraformaldehyde.The expression condition of NSE,OMP were examined by immunohistochemical method in the different embryomic stages from 13th day to the 21th day.TUNEL was applied to detect apoptotic cells of olfactory epithelium in the different embryomic stages,the apoptotic index was expressed for x±s.The statistic data of the apoptotic index was analyzed by Kruskal-Wallis test(P＜0.05)using the software systat version 10.0.Result:In embryonic 13th day,There were positive cells of NSE in the olfactory epitheliu.From E13 to E21,The positive cell of NSE increased and gradually stabilized.Otherwise,the positive cells of OMP were observed since E14 and the quantity was few.In E13 there was no the OMP-positive cell in olfactory epithelium.The noticeable increasing in number of OMP-positive neurons was observed in the olfactory epithelium with embryonic development.In E17,the quantity of OMP-positive neurons reached the peak.Afterward,quantity of OMP-positive neurons reached stabilization.The apoptotic process in the olfactory epithelium was observed form E13 to E21.In E13,the number of apoptotic cells was few and the AI was low.In E14,the number of apoptotic cells increased.A massive wave of cell death was evidenced in E16 and finally decreased to low levels at E19.Afterward,few apoptotic figures were observed and the apoptotic index in E20,E21 was very low.The apoptotic index of E16 was obviously defferentance from others by Kruskal-Wallis test(P＜0.05).The defferentance of the AI between the others was not obvious(P＜0.05).Conclusion:Olfactory receptor neurns of mousefetuses has begun since E13.But in this differentiated stage,the olfactory receptor neurns are immature.In E14,the mature Olfactory receptor neurns emerge and the quantity is few.With the increasing of embryonic days,the number of the mature Olfactory receptor neurns increases.To E21,the olfactory epithelium has been mature.The apoptotic process takes part in the development of the mouse olfactory epithelium.The apoptosis is a active process in the development of the mouse olfactory epithelium and the effect of the apoptosis is important.更多还原