分泌性中耳炎豚鼠模型咽鼓管粘膜内水通道蛋白1,5的表达变化及其作用机制初探

发布时间：2018-04-20 22:23

本文选题：分泌性中耳炎 + 水通道蛋白　；参考：《东南大学》2016年硕士论文

【摘要】：目的：探究模型豚鼠在分泌性中耳炎(Otitis media with effusion, OME)自然转归过程中,咽鼓管黏膜上皮内AQP1、AQP5的表达变化情况,试图从新的角度探讨AQP参与调控中耳液体平衡的机制。方法：健康雄性豚鼠35只,采用左侧咽鼓管阻塞法建立可逆性OME豚鼠模型,耳内镜下观察并记录造模成功豚鼠的鼓膜形态及鼓室积液情况,将造模成功的豚鼠随机分为“术后3d组”、“术后7d组”、“术后14d组”和“术后28d组”四个实验组,分别于术后第3天、第7天、第14天和第28天处死、采集标本。同时取5只健康雄性豚鼠的左侧咽鼓管组织作为对照。苏木素-伊红(HE)染色观察OME豚鼠咽鼓管黏膜的组织学形态变化,免疫组织化学染色观察AQP1、AQP5在OME豚鼠模型咽鼓管黏膜上皮内的表达部位,蛋白免疫印迹法检测各组中AQP1,5的表达量是否有差异。结果：27只实验豚鼠造模成功,术后每三天耳内镜下观察并记录鼓膜形态及鼓室积液情况,将中耳感染及死亡的豚鼠排除实验队列,最终“术后3d组”、“术后7d组”、“术后14d组”和“术后28d组”符合实验要求予以纳入的实验豚鼠例数为6例、5例、4例、3例。HE染色观察到对照组的咽鼓管黏膜纤毛排列整齐,形态完整；而“术后3d组”和“术后7d组”的咽鼓管黏膜出现纤毛紊乱、坏死、缺失,上皮层及固有层水肿,混合性腺体病理性增生等形态学改变。随着疾病的自动转归,在“术后14d组”和“术后28d组”中可以观察到咽鼓管黏膜的纤毛形态逐渐恢复,黏膜水肿较前减轻。免疫组织化学染色观察到AQP1表达于咽鼓管黏膜下的成纤维细胞以及血管内皮细胞；AQP5表达于咽鼓管黏膜的纤毛细胞顶部以及混合性腺体。蛋白免疫印迹实验显示AQP1与AQP5的表达量在“术后3d组”出现了显著降低,“术后7d组”维持在较低水平,而在“术后14d组”出现了表达上升,“术后28d组”中的表达则进一步增加。结论：1.AQP1和AQP5在豚鼠中耳咽鼓管黏膜内存在表达,其中AQP1表达于咽鼓管黏膜下的成纤维细胞以及血管内皮细胞；AQP5表达于咽鼓管黏膜的纤毛细胞顶部以及混合性腺体。2.咽鼓管黏膜内的AQP1和AQP5在OME发病以及自然转归的过程中出现了表达明显下降随后缓慢上升的变化,并且AQP1和AQP5的表达水平与咽鼓管黏膜的受损程度存在关联。3.咽鼓管黏膜内的AQP1和AQP5对于维持咽鼓管黏膜的完整性和黏液纤毛系统的正常运作具有重要意义,并可能通过该途径参与了中耳液体平衡的调节以及OME的发生、发展。
[Abstract]:Objective: To explore the changes in the expression of AQP1 and AQP5 in the epithelia of the eustachian tube during the natural outcome of Otitis media with effusion (OME) in the model guinea pig, and to explore the mechanism of AQP involved in regulating the liquid balance of the middle ear from a new angle. Methods: 35 healthy male guinea pigs were established by the left eustachian tube obstruction method. In a reversible OME guinea pig model, the tympanic membrane morphology and tympanic cavity effusion were observed and recorded by endoscopy. The guinea pigs were randomly divided into four groups, "postoperative 3D group", "postoperative 7d group", "postoperative 14d group" and "postoperative 28d group", respectively, to be killed at third days, seventh days, fourteenth days and twenty-eighth days after operation respectively. At the same time, the left eustachian tube tissue of 5 healthy male guinea pigs was taken as control. The histological changes of the eustachian tube mucosa of OME guinea pigs were observed by hematoxylin eosin (HE) staining. The expression of AQP1, AQP5 in the mucosa of the eustachian tube of OME guinea pig was observed by immunohistochemical staining, and AQP1,5 was detected by protein immunoblotting in the OME guinea pig model. Results: 27 experimental guinea pigs were made successfully. The morphology of the tympanic membrane and the effusion of tympanic cavity were recorded every three days after ear endoscopy. The middle ear infection and the dead guinea pigs were excluded from the experimental cohort, and the final "postoperative 3D group", "postoperative 7d group", "postoperative 14d group" and "postoperative 28d group" were accorded with the experimental requirements. The number of experimental guinea pigs included 6 cases, 5 cases, 4 cases. 3 cases of.HE staining showed that the mucociliary mucosa of the eustachian tube in the control group was neatly arranged, and the morphology of the eustachian tube in the post operation 3D group and the 7d group after operation appeared ciliary disorder, necrosis, deletion, epithelium and lamina edema, and the pathological hyperplasia of mixed gland. In the "postoperative 14d group" and "postoperative 28d group", the cilium morphology of the eustachian tube was gradually recovered and the edema of the mucous membrane decreased. Immunohistochemical staining was used to observe the fibroblasts and intravascular cells expressed under the mucosa of the eustachian tube by immunohistochemical staining. AQP5 was expressed in the cilium of the eustachian tube mucosa. The expression of AQP1 and AQP5 showed a significant decrease in the "postoperative 3D" group. "Post operation 7d group" was maintained at a lower level, while the expression in "postoperative 14d group" increased, and the expression in group 28d was further increased. Conclusion: 1.AQP1 and AQP5 are in guinea pigs. The expression of AQP1 was expressed in the mucous membrane of the eustachian tube, in which the fibroblasts and vascular endothelial cells were expressed under the mucous membrane of the eustachian tube; the AQP1 and AQP5 expressed in the ciliated cells of the eustachian tube and the AQP1 and AQP5 in the mixed.2. eustachian tube mucosa showed a significant decline in the expression of the OME and the natural outcome of the eustachian mucosa. The expression level of AQP1 and AQP5 and the damage degree of the eustachian tube mucosa are associated with the AQP1 and AQP5 in the.3. eustachian tube mucosa. It is important to maintain the integrity of the eustachian tube mucosa and the normal operation of the mucociliary system, and may be involved in the regulation of liquid balance in the middle ear and OME. Happen, develop.

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R764.21;R-332

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