大鼠急性鼻—鼻窦炎模型嗅鞘细胞变化的实验观察

发布时间：2018-03-02 22:06

本文选题：急性鼻-鼻窦炎　切入点：肺炎链球菌　出处：《南昌大学》2011年硕士论文　论文类型：学位论文

【摘要】：第一部分大鼠急性鼻一鼻窦炎模型的建立目的：探讨运用Merocel止血棉和肺炎链球菌制作大鼠急性鼻—鼻窦炎模型。方法：正常SD大鼠50只,随机分为实验组(40只)和对照组(10只)。实验组大鼠右侧鼻腔均置入条棒状Merocel止血棉,同时滴加0.1m1肺炎链球菌菌液于止血棉上；对照组大鼠右侧鼻腔单纯滴入等量生理盐水。实验组大鼠分别于接种细菌后1、2、3、4周处死10只；对照组大鼠均于第1周处死。麻醉、断头、剔除鼻面部软组织,取包含鼻腔鼻窦的组织,4%多聚甲醛固定、10%EDTA脱钙、酒精梯度脱水、石蜡包埋、切片、HE染色观察鼻腔鼻窦炎性反应情况。结果：实验组大鼠鼻腔鼻窦黏膜均有不同程度的炎症细胞侵润及黏膜上皮变薄,第1周炎症反应出现,第2周炎症反应最重,第3周炎症较前减轻,第4周最轻,基本恢复正常,对照组大鼠鼻腔鼻窦黏膜没有炎症反应。结论：运用Merocel止血棉作为肺炎链球菌的载体可建立较其他方法更为稳定便捷的大鼠急性鼻—鼻窦炎模型,为鼻—鼻窦炎的研究提供了可靠的实验基础。第二部分嗅觉功能的检测目的：探讨运用埋藏食物小球实验(buffed food pellet test, BFPT)检测大鼠嗅觉功能的可行性,并分析嗅觉功能的变化情况。方法：正常SD大鼠100只,随机分为实验组(80只)和对照组(20只)。在建立上述大鼠急性鼻—鼻窦炎模型的基础上,采用Nathan等提出的行为学方法一埋藏食物小球法(buffed food pellet test, BFPT)检测各组大鼠的嗅觉功能,并以林静等提出的超过300s未找到食物小球作为大鼠嗅觉障碍的参考标准。实验组大鼠分别于造模后的第1、2、3、4周检测嗅觉功能,每次检测20只(检测之前抽出大鼠右侧鼻腔内填塞的Merocel止血棉),对照组大鼠于第1周检测。记录每只大鼠从放入笼内到找到食物小球(双前肢抱住或牙齿咬住食物小球)的时间。每只大鼠测试两次,分别在受试当天的上午和下午进行测试,间隔6个小时。两次结果取平均值,并进行统计学分析。结果：各实验组大鼠寻找到食物小球的时间均较对照组为长,以第2周最长。并且前三周均超过300s,即均伴有不同程度的嗅觉功能障碍。第4周尽管低于300s,但仍较对照组长。差别均具统计学意义(P0.01)。结论：埋藏食物小球法与病理组织学检查相结合,增加了实验的客观性,可作为评估动物嗅觉功能的一种方法。第三部分成熟嗅感觉神经元变化的实验观察目的：观察大鼠急性鼻一鼻窦炎模型中成熟嗅感觉神经元的变化情况。方法：在上述建立大鼠急性鼻一鼻窦炎动物模型的基础上,用免疫荧光标记法观察嗅黏膜上皮中嗅感觉神经元的变化情况。实验组大鼠分别于接种细菌后1、2、3、4周处死20只；对照组大鼠均于第1周处死。麻醉、断头、剔除鼻面部软组织,于4%多聚甲醛中取包含鼻中隔黏膜的组织,依次进行固定、脱水、包埋、制作冰冻切片、顺序标号。采用抗嗅觉蛋白受体(OMP,标记成熟神经元)观察成熟嗅感觉神经元的变化。结果：对照组大鼠嗅黏膜中下层可见大量排列整齐呈圆形或椭圆形的成熟嗅感觉神经元,固有层亦有散在分布。实验中观察到造模后第1周,成熟嗅感觉神经元出现减少。至第2周炎症反应高峰期,成熟嗅感觉神经元减少,尤为显著。造模后第3周,成熟嗅感觉神经元开始增加。至第4周,成熟嗅感觉神经元较前增多,但是尚未恢复正常神经元的总数。结论：鼻一鼻窦炎导致成熟嗅感觉神经元凋亡增加,并能够诱导嗅感觉神经元再生,但炎性环境下新生嗅感觉神经元的成熟障碍。第四部分嗅鞘细胞变化的实验观察目的：观察大鼠急性鼻一鼻窦炎模型中嗅鞘细胞的变化情况,以进一步探索嗅鞘细胞在鼻—鼻窦炎嗅觉功能恢复中发挥的作用。方法：在上述建立大鼠急性鼻一鼻窦炎动物模型的基础上,应用免疫荧光双标法观察嗅黏膜上皮中嗅鞘细胞的变化情况。在第三部分所制冰冻切片中,选取两张,采用p75NTR抗体和GFAP抗体观察嗅鞘细胞的变化情况。结果：正常嗅黏膜固有层可见大量排列整齐的呈梭形或圆形的嗅鞘细胞。造模后第1周,嗅上皮变薄,固有层嗅鞘细明显减少,同时嗅上皮偶可见OECs生长。造模后第2周,嗅上皮显著变薄,固有层OECs较上周增多,嗅上皮可见明显增多的OECs生长,多为GFAP单阳性未成熟扁圆形,群集生长。造模后第3周,嗅上皮厚度增加,固有层OECs增多,部分形成集落。嗅上皮仍可见少量OECs生长。造模后第4周,嗅上皮厚度基本恢复正常,固有层嗅鞘细胞基本恢复正常,嗅上皮偶可见OECs生长。结论：,急性鼻—鼻窦炎不仅可导致ORNs凋亡增加,同时OECs亦会出现短时间的减少。但随之OECs出现应激性再生增加,尤以第2周炎症高峰期最为明显,直至炎症消退。提示OECs在急性鼻—鼻窦炎嗅觉障碍的恢复中可能发挥了不可忽视的作用。
[Abstract]:The first part of the rat model of acute rhinosinusitis
Objective: To explore the model of acute rhinosinusitis in rats by using Merocel hemostat and Streptococcus pneumoniae.
Methods: 50 SD rats were randomly divided into experimental group (40 rats) and control group (10 rats). Rats in the experimental group were implanted into the right nasal rod dropping the Merocel sponge, 0.1m1 of Streptococcus pneumoniae bacteria on hemostatic cotton; the control group on the right side of rat nasal drops the same amount of pure life saline. Experimental group rats were killed 10 weeks after inoculation of bacteria 1,2,3,4; the control group rats were killed in first weeks. Anesthesia, decapitated, removing nasal facial soft tissue, the nasal tissue, 4% paraformaldehyde, 10%EDTA decalcified, dehydrated with gradient ethanol, paraffin embedded, sliced. HE staining was used to observe the nasal inflammation reaction.
Results: the nasal mucosa of rats in experimental group were significantly different degrees of infiltration of inflammatory cells and epithelial thinning, inflammatory reaction appeared first weeks second weeks, the most important inflammatory response, third weeks before the fourth week of inflammation is reduced, the light returned to normal, the control group of nasal mucosa in rats without inflammation.
Conclusion: the use of Merocel hemostatic cotton as carrier of Streptococcus pneumoniae can establish a more stable and convenient model of acute rhinosinusitis in rats than other methods. It provides a reliable experimental basis for the research of rhinosinusitis.
Detection of olfactory function in the second part
Objective: To explore the feasibility of buffed food pellet test (BFPT) in detecting olfactory function in rats and analyze the change of olfactory function.
Methods: 100 SD rats were randomly divided into experimental group (80 rats) and control group (20 rats). Based on the establishment of the rat acute nose - sinusitis model, proposed by Nathan and the behavior of a buried food pellet method methodology (buffed food pellet test, BFPT) were measured the rat olfactory function, and more than to put forward by Lin Jing 300s did not find a food pellet as olfactory dysfunction in rats. The rats of the experimental group reference standard respectively after modeling 1,2,3,4 weeks of detection of olfactory function, each test 20 (before testing out the rat right nasal packing Merocel hemostatic cotton). The rats in the control group in the first week test. Records of each rat from into the cage to find food pellets (double teeth or hold forelimb food pellet) time. Each rat was tested two times respectively in the subjects on the day of the morning and afternoon test interval of 6 hours. The average value of the two results was taken and the statistical analysis was carried out.
Results: the experimental rats to find the food pellet time were higher than those in control group, second weeks and three weeks before the longest. More than 300s, which were accompanied by varying degrees of olfactory dysfunction. Fourth weeks despite lower than 300s, but still than in the control group. The difference was statistically significant (P0.01).
Conclusion: the combination of buried food ball method and histopathological examination can increase the objectivity of the experiment, and can be used as a method to evaluate the olfactory function of animals.
Experimental observation on the changes of the third part of the mature olfactory sensory neurons
Objective: To observe the changes of the mature olfactory sensory neurons in the rat model of acute rhinosinusitis.
Methods: in the basis of acute rat nasal sinusitis animal model, to observe the changes of olfactory epithelium of olfactory sensory neurons by immunofluorescence method. The experimental group rats were killed 20 weeks after inoculation of bacteria 1,2,3,4; the rats in control group were dead at first weeks. Anesthesia, decapitation, culling nasal and facial soft tissue in 4% paraformaldehyde in the nasal mucosa tissue were fixed, dehydrated, embedded, frozen sections were made, sequence labeling. The anti olfactory receptor protein (OMP, markers of mature neurons) observed in mature olfactory sensory neurons.
Results: in the control group in the rat olfactory mucosa layer shows a large number of rows in the mature olfactory sensory neurons round or oval, are scattered in the lamina propria. Observed first weeks after the experiment, the mature olfactory sensory neurons decreased to second weeks. The inflammatory response peak, mature olfactory sensory neurons decreased significantly at third weeks after modeling, the mature olfactory sensory neurons began to increase. At fourth weeks, the mature olfactory sensory neurons was increased, but the total number of neurons have not yet returned to normal.
Conclusion: rhinosinusitis can increase the apoptosis of mature olfactory sensory neurons and induce the regeneration of olfactory sensory neurons, but the maturation of new olfactory sensory neurons in inflammatory environment.
Experimental observation on the changes of olfactory ensheathing cells in the fourth part
Objective: To observe the changes of olfactory ensheathing cells in acute rhinosinusitis model in rats, so as to further explore the role of olfactory ensheathing cells in the recovery of olfactory function in rhinosinusitis.
Methods: in the basis of acute rat nasal sinusitis animal model, double immunofluorescence method was used to observe the changes of olfactory ensheathing cells of the olfactory epithelium. In the third part of the frozen section, select two, using p75NTR and GFAP antibody to observe the changes of olfactory ensheathing cells.
Results: the normal olfactory mucosa lamina propria in large fusiform or round arrangement of olfactory ensheathing cells were neat. First weeks after modeling, the thinning of the olfactory epithelium, lamina propria of olfactory ensheathing fine was reduced, while the olfactory epithelium even visible growth of OECs. Second weeks after modeling, significant thinning of the olfactory epithelium, lamina propria OECs last week increased, the olfactory epithelium increased significantly OECs visible growth for GFAP single positive immature oblate, cluster growth. Third weeks after modeling, the increase of the thickness of the olfactory epithelium, lamina propria OECs increased, part of the colony formation. The olfactory epithelium was still a small growth of OECs. Fourth weeks after modeling, the thickness of the olfactory epithelium basic return to normal, the lamina propria of olfactory ensheathing cells returned to normal, the olfactory epithelium showed occasional OECs growth.
Conclusion: acute rhinosinusitis can not only lead to the apoptosis of ORNs, while OECs will also appear to reduce short time. But with OECs stress of regeneration increased, especially in the second week peak of inflammation was most obvious, until the inflammation subsided. It suggested that OECs might play a noticeable role in olfactory dysfunction in acute rhinosinusitis the recovery.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R765.21

【引证文献】