糖皮质激素对分泌性中耳炎模型水通道蛋白1表达的调控

发布时间：2019-04-03 12:55

【摘要】：目的 1、探索单纯咽鼓管阻塞建立分泌性中耳炎动物模型的方法，为分泌性中耳炎急性期的相关实验研究提供一种理想的动物模型。 2、探寻水通道蛋白1在正常和分泌性中耳炎豚鼠中耳腔的表达和定位，以及在分泌性中耳炎病程中的表达变化，同时在蛋白水平观察糖皮质激素对水通道蛋白1表达的调控作用。方法 1、健康雄性豚鼠22只，左侧为实验耳，右侧为对照耳，经软腭切开膨胀海绵阻塞左侧咽鼓管咽口建立可逆性分泌性中耳炎动物模型，耳内镜下观察记录鼓膜形态和鼓室积液情况，分别于术后第7天、第14天及第21天各获取6只造模成功的豚鼠听泡，探查术后膨胀海绵留置情况，HE染色观察急性分泌性中耳炎病程中中耳黏膜的病理改变。 2、用免疫组织化学的方法观察正常和分泌性中耳炎豚鼠中耳腔水通道蛋白1的表达和分布情况。 3、造模成功的豚鼠20只，随机分为Dexa组（治疗组）和OME组（非治疗组），，每组10只。Dexa组术后第7天起，每天腹腔注射地塞米松5mg/kg，持续7天。术后第14天处死2组采集标本。同时取5只正常豚鼠的双侧听泡做为NC组（正常对照）。用免疫印迹方法在蛋白水平分别检测3组水通道蛋白1表达量且比较有无差异。结果 1、造模组22耳中20耳分别于术后第3～7天出现鼓室浆液性渗出，造模成功率为90.9%，病理改变表现为鼓室黏膜增厚，黏膜下血管扩张，淋巴细胞浸润，近咽鼓管鼓室口部纤毛低矮，数量减少甚至脱落。术后14 d膨胀海绵已基本排出，14～18 d中耳积液消失，同时组织形态逐渐恢复正常。病程中对侧对照耳鼓室无积液，亦无明显病理改变。 2、水通道蛋白1在正常和分泌性中耳炎豚鼠中耳腔黏膜内有明确表达，中耳腔黏膜下层的毛细血管内皮和成纤维细胞均有水通道蛋白1分布。分泌性中耳炎豚鼠中耳腔的表达更为显著。 3、3组的豚鼠中耳腔黏膜中，抗水通道蛋白1抗体在1：3000的工作浓度下识别出28kDa条带。独立样本t检验两两比较提示3组的蛋白表达量皆有差异。NC组的蛋白表达水平高于另外2组，Dexa组的蛋白表达水平显著高于OME组(t=2.733，P0.05)。结论 1、经软腭切开膨胀海绵咽鼓管阻塞法可成功建立豚鼠急性分泌性中耳炎模型，该分泌性中耳炎可于膨胀海绵自行排出后2周逐渐自愈。 2、水通道蛋白1在豚鼠中耳腔黏膜有表达，参与中耳腔的液体平衡过程。 3、水通道蛋白1在分泌性中耳炎自然转归过程中表达水平发生变化。糖皮质激素可上调水通道蛋白1的表达，并且可能是通过此机制协同实现对分泌性中耳炎治疗作用。
[Abstract]:Aim 1. To explore the method of establishing the animal model of secretory otitis media by simple eustachian tube obstruction, and to provide an ideal animal model for the experimental study of secretory otitis media in the acute stage. 2. To explore the expression and localization of aquaporin-1 in the middle ear cavity of guinea pigs with normal and secretory otitis media, and the changes of expression of aquaporin-1 in the course of secretory otitis media. The effects of glucocorticoid on the expression of aquaporin 1 were also observed at the protein level. Methods 1. Twenty-two healthy male guinea pigs with experimental ears on the left and control ears on the right were used to establish reversible secretory otitis media model through soft palate incision and swelled sponge blocking the pharynx mouth of the left pharynx tube, and the animal model of reversible secretory otitis media was established. The morphology of tympanic membrane and effusion of tympanum were observed and recorded under endoscope. Six guinea pig auditory vesicles were obtained on the 7th day, 14th day and 21st day after operation, respectively, and the indwelling condition of swelling sponge after operation was explored. HE staining was used to observe the pathological changes of ear mucosa in the course of acute secretory otitis media. 2. The expression and distribution of aquaporin 1 in the middle ear of guinea pigs with normal and secretory otitis media were observed by immunohistochemical method. 3. 20 guinea pigs were randomly divided into two groups: Dexa group (treatment group) and OME group (non-treatment group), 10 guinea pigs in each group. Dexa group received intraperitoneal injection of dexamethasone 5 mg / kg daily for 7 days from the 7th day after operation, and 10 guinea pigs in each group received intraperitoneal injection of dexamethasone (5 mg / kg) per day for 7 days. Two groups were sacrificed on the 14th day after operation. At the same time, the bilateral auditory vesicles of 5 normal guinea pigs were taken as NC group (normal control). The expression of aquaporin-1 in the three groups was detected by Western blot at the protein level and there was no difference in the expression of aquaporin-1. Results 1 in 20 ears of 22 ears in the model group, tympanic serous exudation occurred on the 3rd to 7th day after operation, and the success rate of the model was 90.9%. The pathological changes were as follows: thickening of tympanic mucosa, dilation of submucosal vessels and infiltration of lymphocytes. The cilia of the tympanic orifice near the eustachian tube was low and the number decreased or even fell off. The swelling sponge was basically discharged on the 14th day after operation, the middle ear effusion disappeared on the 14th day after operation, and the histologic morphology gradually returned to normal at the same time. There was no effusion in the contralateral tympanic chamber and no obvious pathological changes in the course of the disease. 2. Aquaporin-1 was clearly expressed in the middle ear mucosa of normal and secretory otitis media guinea pigs, and aquaporin-1 distribution was found in both capillary endothelium and fibroblasts of the submucosa of the middle ear cavity. The expression of secretory otitis media in the middle ear cavity of guinea pigs was more significant. 3. Anti-aquaporin-1 antibody detected 28kDa bands in the middle ear mucosa of guinea pigs in 3 groups at the working concentration of 1? 3 000. The independent sample t-test showed that the protein expression level in NC group was higher than that in the other two groups, and the protein expression level in Dexa group was significantly higher than that in OME group (t = 2.773, P0.05). The expression level of protein in Dexa group was significantly higher than that in OME group (t = 2.733, P0.05). Conclusion 1. Acute secretory otitis media model of guinea pigs can be successfully established by soft palate incision and swelling sponge eustachian tubule obstruction. The secretory otitis media can heal itself 2 weeks after expandable sponge. 2 weeks after the swelling sponge is discharged by itself, the model of secretory otitis media can be established successfully. 2. Aquaporin-1 was expressed in the middle ear mucosa of guinea pigs and was involved in the process of fluid balance in the middle ear cavity of guinea pigs. 3. The expression level of aquaporin-1 in secretory otitis media was changed during the course of spontaneous prognosis. Glucocorticoid can up-regulate the expression of aquaporin-1, and may be a synergistic mechanism for the treatment of secretory otitis media.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764

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